Neisseria meningitidis compositions and methods thereof

ABSTRACT

In one aspect, the invention relates to a composition including a first polypeptide having the sequence set forth in SEQ ID NO: 1 and a second polypeptide having the sequence set forth in SEQ ID NO: 2. In one embodiment, the composition includes about 120 μg/ml of a first polypeptide including the amino acid sequence set forth in SEQ ID NO: 1, 120 μg/ml of a second polypeptide including the amino acid sequence set forth in SEQ ID NO: 2, about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8 molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/ml aluminum, about 10 mM histidine, and about 150 mM sodium chloride. In one embodiment, a dose of the composition is about 0.5 ml in total volume. In one embodiment, two-doses of the composition induce a bactericidal titer against diverse heterologous subfamily A and subfamily B strains in a human.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 61/875,068, filed Sep. 8, 2013, U.S. provisionalpatent application Ser. No. 61/926,717, filed Jan. 13, 2014, and U.S.provisional patent application Ser. No. 61/989,432, filed May 6, 2014,which are hereby incorporated herein by reference in their respectiveentirety.

FIELD OF THE INVENTION

The present invention relates to Neisseria meningitidis compositions andmethods thereof.

BACKGROUND OF THE INVENTION

Neisseria meningitidis is a Gram-negative encapsulated bacterium thatcan cause sepsis, meningitis, and death. N. meningitidis can beclassified into at least 12 serogroups (including serogroups A, B, C,29E, H, I, K, L, W-135, X, Y and Z) based on chemically andantigenically distinctive polysaccharide capsules. Strains with five ofthe serogroups (A, B, C, Y, and W135) are responsible for the majorityof disease.

Meningococcal meningitis is a devastating disease that can kill childrenand young adults within hours despite the availability of antibiotics.There is a need for improved immunogenic compositions againstmeningococcal serogroups A, B, C, Y, and W135 and/or X.

Currently, a cross-protective vaccine or composition effective against awide range of MnB isolates is not yet commercially available. Forexample, published results-to-date relating to a licensedmulti-component composition for protection against serogroup B diseasehas not demonstrated a direct bactericidal immune response againstmultiple strains expressing heterologous LP2086 (fHBP) variants, atleast in adolescents. At most, published results-to-date relating to themulti-component composition for protection against serogroup B diseaseappear to show immunogenicity against LP2086 (fHBP) variants that arehomologous to the LP2086 (fHBP) variant in the multi-componentcomposition. Accordingly, a cross-protective vaccine or compositioneffective against diverse MnB isolates is needed as is determiningreal-world vaccine coverage against a panel of diverse or heterologousmeningococcal strains (e.g., representing different geographicalregions).

SUMMARY OF THE INVENTION

To meet these and other needs, the present invention relates toNeisseria meningitidis compositions and methods thereof.

In one aspect, the invention relates to a composition including about120 μg/ml of a first lipidated polypeptide including the amino acidsequence set forth in SEQ ID NO: 1, 120 μg/ml of a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2,about 2.8 molar ratio polysorbate-80 to the first polypeptide, about 2.8molar ratio polysorbate-80 to the second polypeptide, about 0.5 mg/mlaluminum, about 10 mM histidine, and about 150 mM sodium chloride. Inone embodiment, the first dose is about 0.5 ml in total volume. In oneembodiment, the composition induces a bactericidal immune responseagainst N. meningitidis serogroup B. In one embodiment, the compositioninduces a bactericidal immune response against N. meningitidis serogroupA, C, 29E, H, I, K, L, W-135, X, Y or Z. In one embodiment, thecomposition does not further include a polypeptide having less than 100%sequence identity to SEQ ID NO: 1. In one embodiment, the compositiondoes not further include a polypeptide having less than 100% sequenceidentity to SEQ ID NO: 2. In one embodiment, the first polypeptide has atotal of 258 amino acids. In one embodiment, the second polypeptide hasa total of 261 amino acids. In one embodiment, the composition induces abactericidal titer of serum immunoglobulin that is at least 2-foldhigher in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, wherein the increase in bactericidal titer is measured underidentical conditions in a serum bactericidal assay using humancomplement. In one embodiment, the first lipidated polypeptide consistsof the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment,the second lipidated polypeptide consists of the amino acid sequence setforth in SEQ ID NO: 2.

In another aspect, the invention relates to a method of inducing animmune response against Neisseria meningitidis in a human. The methodincludes administering to the human a first dose and a second dose of aneffective amount of a composition, said composition including 120 μg/mlof a first lipidated polypeptide including the amino acid sequence setforth in SEQ ID NO: 1, 120 μg/ml of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molarratio polysorbate-80 to the first polypeptide, 2.8 molar ratiopolysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride. In one embodiment, a dose of thecomposition has a total volume of 0.5 ml. In one embodiment, the humanis administered at most two doses of the composition. In one embodiment,the human is not further administered a booster dose of the composition.In one embodiment, the human is administered a third dose of thecomposition. In one embodiment, the human is not further administered abooster dose of the composition after the third dose. In one embodiment,the human is not further administered a fourth dose of the composition.In one embodiment, the third dose is administered to the human within aperiod of about 6 months after the first dose. In one embodiment, thesecond dose is administered at least 30 days after the first dose. Inone embodiment, the method further includes administering a third doseof the composition, wherein the third dose is administered at least 90days after the second dose. In one embodiment, the composition induces abactericidal titer of serum immunoglobulin that is at least 2-foldhigher in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement. In one embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily A strainthat is heterologous to a N. meningitidis strain expressing A05. In oneembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to a N.meningitidis strain expressing B01. In one embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain that is heterologous to N. meningitidis strain M98250771. Inone embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to N.meningitidis strain CDC1127. In a preferred embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyB strain that is heterologous to N. meningitidis strain CDC1573. In oneembodiment, the first polypeptide has a total of 258 amino acids. In oneembodiment, the second polypeptide has a total of 261 amino acids. Inone embodiment, the first lipidated polypeptide consists of the aminoacid sequence set forth in SEQ ID NO: 1. In one embodiment, the secondlipidated polypeptide consists of the amino acid sequence set forth inSEQ ID NO: 2.

In another aspect, the invention relates to a composition that includes60 μg of a first lipidated polypeptide including the amino acid sequenceset forth in SEQ ID NO: 1, 60 μg of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2, 2.8 molarratio polysorbate-80 to the first polypeptide, 2.8 molar ratiopolysorbate-80 to the second polypeptide, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride, wherein the composition has atotal volume of about 0.5 ml. In one embodiment, the composition inducesa bactericidal immune response against a N. meningitidis serogroup Bsubfamily A strain that is heterologous to a N. meningitidis strainexpressing A05. In one embodiment, the composition induces abactericidal immune response against a N. meningitidis serogroup Bsubfamily B strain that is heterologous to a N. meningitidis strainexpressing B01. In one embodiment, the composition induces abactericidal titer of serum immunoglobulin that is at least 2-foldhigher in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement. In one embodiment, the composition doesnot further include a polypeptide having less than 100% sequenceidentity to SEQ ID NO: 1. In one embodiment, the composition does notfurther include a polypeptide having less than 100% sequence identity toSEQ ID NO: 2. In one embodiment, the first polypeptide has a total of258 amino acids. In one embodiment, the second polypeptide has a totalof 261 amino acids. In one embodiment, the first lipidated polypeptideconsists of the amino acid sequence set forth in SEQ ID NO: 1. In oneembodiment, the second lipidated polypeptide consists of the amino acidsequence set forth in SEQ ID NO: 2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Proportion of Subjects Achieving hSBA Titers ≧LLOQ. hSBA=serumbactericidal assay using human complement; LLOQ=lower limit ofquantitation.

FIG. 2—Percentage of subjects achieving 4× rise in hSBA titers toPrinceton University Outbreak Strains and UCSB Outbreak Strains ofIndividual Human Subjects Following Immunization With rLP2086 (StudyB1971012—described in Example 5, Example 6). Serum samples from ninehuman subjects immunized with bivalent rLP2086 in clinical studyB1971012 were evaluated in exploratory hSBAs using MnB outbreak strainsfrom Princeton University and from UCSB. See Example 9.

SEQUENCE IDENTIFIERS

SEQ ID NO: 1 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant A05 polypeptide antigen.

SEQ ID NO: 2 sets forth the amino acid sequence for a recombinant N.meningitidis, serogroup B, 2086 variant B01 polypeptide antigen.

SEQ ID NO: 3 sets forth the amino acid residues at positions 1-4 of SEQID NO: 1 and SEQ ID NO: 2.

SEQ ID NO: 4 sets forth the amino acid sequence of the N-terminus of arecombinant Neisserial Subfamily A LP2086 polypeptide (rLP2086) (A05)polypeptide antigen.

SEQ ID NO: 5 sets forth the amino acid sequence of the N-terminus ofNeisserial Subfamily A LP2086 M98250771 polypeptide (A05) polypeptideantigen.

SEQ ID NO: 6 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B153.

SEQ ID NO: 7 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A04.

SEQ ID NO: 8 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A05

SEQ ID NO: 9 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A12.

SEQ ID NO: 10 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A22.

SEQ ID NO: 11 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B02.

SEQ ID NO: 12 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B03.

SEQ ID NO: 13 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B09.

SEQ ID NO: 14 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B22.

SEQ ID NO: 15 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B24.

SEQ ID NO: 16 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B44.

SEQ ID NO: 17 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B16.

SEQ ID NO: 18 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A07.

SEQ ID NO: 19 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A19.

SEQ ID NO: 20 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A06.

SEQ ID NO: 21 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A15.

SEQ ID NO: 22 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant A29.

SEQ ID NO: 23 sets forth the amino acid sequence for N. meningitidis,serogroup B, 2086 variant B15.

DETAILED DESCRIPTION OF THE INVENTION

The inventors surprisingly discovered a composition that includes afirst lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 1 and a second lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 2. The composition has anacceptable safety profile in humans and the composition surprisinglyelicits a broadly cross-reactive bactericidal immune response in humansagainst at least more than two diverse Neisseria meningitidis strains.

The inventors further discovered that a 2-dose administration scheduleand a 3-dose administration schedule surprisingly yielded hSBA titers of≧8 against test strains from N. meningitidis serogroup B, with vaccineheterologous LP2086 (factor H binding protein (fHBP)) subfamilies A andB in a high proportion of human subjects. A 3-dose administrationschedule may provide the broadest protection in humans against diverseMnB clinical strains, when compared to a 2-dose administration schedule.

The inventors also surprisingly discovered that robust immune responsesagainst human papillomavirus and N. meningitidis serogroup B weregenerated after concomitant administration of the rLP2086 compositionand a quadrivalent immunogenic composition against human papillomavirus(HPV4). For example, a concomitant administration of the rLP2086composition and HPV4 composition generated an immune response at leastagainst N. meningitidis serogroup B test strains expressing fHBPs thatare heterologous to those fHBPs in the rLP2086 composition. Suchheterologous test strains include wild-type N. meningitidis serogroup Bstrains that express A22 fHBP, A56 fHBP, B24 fHBP, or B44 fHBP, whichare each heterologous to the fHBPs in the rLP2086 composition. SeeWO/2012/032489, WO/2013/132452, US patent publication numberUS20120093852, and US patent publication number US20130243807, whichdescribe variant fHBP proteins, including A22 fHBP, A56 fHBP, B24 fHBP,and B44 fHBP, among others. These references are each incorporated byreference in their entirety. The concomitant administration alsosurprisingly generated an immune response at least against HPV types 6,11, 16, and/or 18. The immune responses against the HPV types afterconcomitant administration of the rLP2086 composition and the HPV4composition were noninferior when compared to the immune responsegenerated by an administration of the HPV4 composition in the absence ofthe rLP2086 composition.

In addition, the inventors surprisingly discovered that robust immuneresponses against diphtheria, tetanus, pertussis and poliomyelitis andN. meningitidis serogroup B were generated after concomitantadministration of the rLP2086 composition and an immunogenic compositionagainst diphtheria, tetanus, pertussis and poliomyelitis. For example, aconcomitant administration of the rLP2086 composition and REPEVAXcomposition generated an immune response at least against N.meningitidis serogroup B test strains expressing fHBPs that areheterologous to those fHBPs in the rLP2086 composition. The concomitantadministration also surprisingly generated an immune response at leastagainst the 9 antigens in REPEVAX: diphtheria, tetanus, pertussistoxoid, pertussis filamentous hemagglutinin, pertussis pertactin,pertussis fimbrial agglutinogens type 2+3, poliovirus type 1, poliovirustype 2, poliovirus type 3. The immune responses against the REPEVAXantigens after concomitant administration of the rLP2086 composition andthe REPEVAX composition were noninferior when compared to the immuneresponse generated by an administration of the REPEVAX composition inthe absence of the rLP2086 composition.

Moreover, the inventors surprisingly discovered that the rLP2086composition induces a bactericidal immune response against an ST409 N.meningitidis strain that expresses the fHBP B153 variant. For example,the strain expressing the fHBP B153 variant was found to be susceptibleto killing when contacted with human bivalent rLP2086 composition immunesera, in a serum bactericidal assay using human complement (hSBA).

Composition and Vaccine

In one aspect, the invention relates to a composition against Neisseriameningitidis. The composition includes a first lipidated polypeptidehaving the amino acid sequence set forth in SEQ ID NO: 1, and a secondlipidated polypeptide having the amino acid sequence set forth in SEQ IDNO: 2.

The inventors surprisingly discovered a single N. meningitidispolypeptide component that induces an effective broadly protectiveimmune response against multiple strains of N. meningitidis serogroup B.Accordingly, in one embodiment, the composition does not include afusion protein. In one embodiment, the composition does not include achimeric protein. In one embodiment, the composition does not include ahybrid protein. In one embodiment, the composition does not furtherinclude a peptide fragment. In another embodiment, the composition doesnot further include a Neisserial polypeptide that is not fHBP. Forexample, in one embodiment, the composition does not include a PorAprotein. In another embodiment, the composition does not include a NadAprotein. In another embodiment, the composition does not further includea Neisserial heparin binding antigen (NHBA). In another embodiment, thecomposition does not further include a Neisserial outer membrane vesicle(OMV). In a preferred embodiment, the composition does not furtherinclude antigens, other than the first polypeptide and the secondpolypeptide.

In another aspect, the inventors surprisingly discovered thatpolypeptide antigens derived from at most two N. meningitidis serogroupB strains induces an effective broadly protective immune responseagainst multiple strains of N. meningitidis serogroup B. Accordingly, inone embodiment, the composition does not further include a polypeptidethat is not derived from N. meningitidis serogroup B subfamily AM98250771 strain and/or N. meningitidis serogroup B subfamily B CDC1573strain.

In one embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 1. Inanother embodiment, the composition does not further include apolypeptide having less than 100% sequence identity to SEQ ID NO: 2. Forexample, the composition does not further include a polypeptide havingless than 100% sequence identity to the full length of SEQ ID NO: 1and/or SEQ ID NO: 2.

In one embodiment, the composition further includes polysorbate-80,aluminum, histidine, and sodium chloride. In one embodiment, thecomposition includes about 60 μg of a first lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1, about 60 μgof a second lipidated polypeptide including the amino acid sequence setforth in SEQ ID NO: 2, 2.8 molar ratio of polysorbate-80 to eachpolypeptide, 0.5 mg aluminum/ml as aluminum phosphate, 10 mM histidine,and 150 mM sodium chloride, wherein the composition preferably has atotal volume of about 0.5 ml.

In another aspect, the composition includes about 120 μg/ml of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1, about 120 μg/ml of a second lipidated polypeptide includingthe amino acid sequence set forth in SEQ ID NO: 2, 2.8 molar ratio ofpolysorbate-80 to each polypeptide, 0.5 mg aluminum/ml as aluminumphosphate, 10 mM histidine, and 150 mM sodium chloride.

In a further aspect, the composition includes a) 60 μg of a firstlipidated polypeptide including the amino acid sequence set forth in SEQID NO: 1; b) 60 μg of a second lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 2; c) 18 μg polysorbate-80; d) 250μg aluminum; e) 780 μg histidine, and; f) 4380 μg sodium chloride.

In an exemplary embodiment, the composition includes about 60 μg of afirst lipidated polypeptide consisting of the amino acid sequence setforth in SEQ ID NO: 1, about 60 μg of a second lipidated polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 2, 2.8molar ratio of polysorbate-80 to first lipidated polypeptide and tosecond lipidated polypeptide, 0.5 mg/ml aluminum phosphate, 10 mMhistidine, and 150 mM sodium chloride, wherein the composition has atotal volume of about 0.5 ml. In the exemplary embodiment, thecomposition is a sterile isotonic buffered liquid suspension. In theexemplary embodiment, the composition has a pH 6.0. In the exemplaryembodiment, the first polypeptide and the second polypeptide areadsorbed to aluminum.

In one embodiment, the composition has a total volume of about 0.5 ml.In one embodiment, a first dose of the composition has a total volume ofabout 0.5 ml. A “first dose” refers to the dose of the composition thatis administered on Day 0. A “second dose” or “third dose” refers to thedose of the composition that is administered subsequently to the firstdose, which may or may not be the same amount as the first dose.

The composition is immunogenic after administration of a first dose to ahuman. In one embodiment, the first dose is about 0.5 ml in totalvolume.

The composition induces a bactericidal titer of serum immunoglobulinthat is at least greater than 1-fold higher, preferably at least 2-foldhigher, in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement (hSBA).

The bactericidal titer or bactericidal immune response is against N.meningitidis serogroup B. In a preferred embodiment, the bactericidaltiter or bactericidal immune response is against a N. meningitidisserogroup B subfamily A strain and against a N. meningitidis serogroup Bsubfamily B strain. Most preferably, the bactericidal titer orbactericidal immune response is at least against N. meningitidisserogroup B, subfamily B, B01 strain.

In one embodiment, the composition induces a bactericidal titer of serumimmunoglobulin that is at least greater than 1-fold, such as, forexample, at least 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold,13-fold, 14-fold, 15-fold, or 16-fold higher in the human afterreceiving a dose of the composition than a bactericidal titer of serumimmunoglobulin in the human prior to receiving said dose, when measuredunder identical conditions in a serum bactericidal assay using humancomplement.

In one embodiment, the composition is an immunogenic composition. In oneembodiment, the composition is an immunogenic composition for a human.In another embodiment, the composition is a vaccine. A “vaccine” refersto a composition that includes an antigen, which contains at least oneepitope that induces an immune response that is specific for thatantigen. The vaccine may be administered directly into the subject bysubcutaneous, oral, oronasal, or intranasal routes of administration.Preferably, the vaccine is administered intramuscularly. In oneembodiment, the composition is a human vaccine. In one embodiment, thecomposition is an immunogenic composition against N. meningitidis.

In one embodiment, the composition is a liquid composition. In apreferred embodiment, the composition is a liquid suspensioncomposition. In another preferred embodiment, the composition is notlyophilized.

First Polypeptide

In one embodiment, the composition includes a first polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 1. In one preferredembodiment, the composition includes about 60 μg of a first polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 1, wherein thecomposition preferably has a total volume of 0.5 ml. In anotherembodiment, the composition includes about 120 μg/ml of a firstpolypeptide including the amino acid sequence set forth in SEQ ID NO: 1.The polypeptide is a modified factor H binding protein (fHBP) from N.meningitidis strain M98250771. A description of fHBP is disclosed inWO2012032489 and US patent publication US 2012/0093852, which are eachincorporated by reference in their entirety. The polypeptide isN-terminally lipidated with three predominant fatty acids C16:0, C16:1,and C18:1 covalently linked at three positions of the polypeptide. Thefirst polypeptide includes a total of 258 amino acids.

The first polypeptide includes two modifications introduced in theN-terminal region of the polypeptide, as compared to the correspondingwild-type sequence from N. meningitidis strain M98250771. A glycine inthe second position is added as a consequence of introducing a cloningsite. A second modification includes the deletion of four amino acids.Accordingly, in one embodiment, the first polypeptide includes a C-G-S-Ssequence (SEQ ID NO: 3) at the N-terminus. See SEQ ID NO: 1, first fouramino acid residues.

The N-terminal differences between the first polypeptide sequence andthe wild-type Neisserial sequence is shown below. Accordingly, in oneembodiment, the first polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, or more amino acid residues of the aminoacid sequence set forth in SEQ ID NO: 1. Preferably, the firstpolypeptide includes at least the first 4, more preferably at least thefirst 6, and most preferably, at least the first 8 amino acid residuesof SEQ ID NO: 1.

Comparison of Predicted N-Terminal Sequences of Recombinant andNeisserial Subfamily A LP2086 Polypeptide

rLP2086 M98250771 (SEQ ID NO: 4) CGSS-----GGGGVAAD Neisserial LP2086M98250771 (SEQ ID NO: 5) C-SSGS-GSGGGGVAAD >A05 (SEQ ID NO: 1)CGSSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVH EIGIAGKQ

In one embodiment, the first polypeptide includes the amino acidsequence set forth in SEQ ID NO: 1. In one embodiment, the firstpolypeptide has a total of 258 amino acids. In one embodiment, the firstpolypeptide does not include an amino acid sequence having less than100% sequence identity to SEQ ID NO: 1. In another embodiment, the firstpolypeptide consists of the amino acid sequence set forth in SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence KDN. See for example, amino acid residues 73-75 of SEQ IDNO: 1. In another embodiment, the first polypeptide includes the aminoacid sequence set forth in SEQ ID NO: 3 at the N-terminus of thepolypeptide. In another embodiment, the first polypeptide includes theamino acid sequence set forth in SEQ ID NO: 4 at the N-terminus of thepolypeptide.

In a preferred embodiment, the first polypeptide is readily expressed ina recombinant host cell using standard techniques known in the art. Inanother preferred embodiment, the first polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 1. In oneembodiment, the first polypeptide includes at least the first 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100 amino acid residues of the amino acid sequence set forthin SEQ ID NO: 1. Preferably, the first polypeptide includes at least thefirst 2, more preferably at least the first 4, and most preferably, atleast the first 8 amino acid residues of SEQ ID NO: 1.

In another embodiment, the first polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 1.

Second Polypeptide

In one embodiment, the composition includes a second polypeptide havingthe amino acid sequence set forth in SEQ ID NO: 2. In one preferredembodiment, the composition includes about 60 μg of a second polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2, wherein thecomposition preferably has a total volume of 0.5 ml. In anotherembodiment, the composition includes 120 μg/ml of a second polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2. Thepolypeptide is a factor H binding protein (fHBP) from N. meningitidisstrain CDC1573. A description of fHBP is disclosed in WO2012032489 andUS patent publication US 2012/0093852, which are each incorporated byreference in their entirety. The polypeptide is N-terminally lipidatedwith three predominant fatty acids C16:0, C16:1, and C18:1 covalentlylinked at three positions of the polypeptide. The second polypeptideincludes a total of 261 amino acids. In one embodiment, the secondpolypeptide includes a C-G-S-S sequence (SEQ ID NO: 3) at theN-terminus. See the first four amino acid residues of SEQ ID NO: 2.

>B01 (SEQ ID NO: 2) CGSSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETAN GIHHIGLAAKQ

In one embodiment, the second polypeptide includes the amino acidsequence set forth in SEQ ID NO: 2. In one embodiment, the secondpolypeptide has a total of 261 amino acids. In one embodiment, thesecond polypeptide consists of the amino acid sequence set forth in SEQID NO: 2. In another embodiment, the second polypeptide does not furtherinclude a polypeptide having less than 100% sequence identity to SEQ IDNO: 2. In a preferred embodiment, the first polypeptide and the secondpolypeptide includes a C-G-S-S(SEQ ID NO: 3) sequence at the N-terminusof the respective polypeptide.

In a preferred embodiment, the second polypeptide is readily expressedin a recombinant host cell using standard techniques known in the art.In another preferred embodiment, the second polypeptide includes abactericidal epitope on the N- and/or C-domain of SEQ ID NO: 2. In oneembodiment, the second polypeptide includes at least the first 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100 amino acid residues of the amino acid sequenceset forth in SEQ ID NO: 2. Preferably, the second polypeptide includesat least the first 2, more preferably at least the first 4, and mostpreferably, at least the first 8 amino acid residues of SEQ ID NO: 2.

In another embodiment, the first polypeptide includes at least the last4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100 amino acid residues of the amino acidsequence set forth in SEQ ID NO: 2.

Polysorbate-80

Polysorbate 80 (PS-80) is a non-ionic surfactant. Accelerated stabilitystudies using an in vitro monoclonal antibody based potency assaydemonstrated instability of the subfamily B protein at higher molarratios of PS-80 to MnB rLP2086 protein in the final formulation. Furtherexperiments with varying ratios of PS-80 have demonstrated that theoptimal molar ratio of PS-80 to MnB rLP2086 protein is approximately2.8±1.4 to retain potency.

The concentration of PS-80 in the composition is dependent on a molarratio of PS-80 to the polypeptide. In one embodiment, the compositionincludes a 2.8±1.4 molar ratio of PS-80 to the first polypeptide and tothe second polypeptide. In one embodiment, the composition includes a2.8±1.1 molar ratio of PS-80 to the first polypeptide and to the secondpolypeptide. In one embodiment, the composition includes at least 1.9,2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3molar ratio of PS-80 to polypeptide. Preferably, the compositionincludes a 2.8 molar ratio of PS-80 to polypeptide.

The PS-80 to polypeptide molar ratio is determined by calculation fromthe measured concentration of PS-80 and the measured total polypeptideconcentration, in which both values are expressed in moles. For example,PS-80 to Protein molar ratio is determined by calculation of themeasured concentration of PS-80 (e.g., by reverse phase high pressureliquid chromatography (RP-HPLC)) to the measured total proteinconcentration (e.g., by ion exchange-high pressure liquid chromatography(IEX-HPLC)) in the final drug substance, where both values are expressedin moles.

A RP-HPLC is used to quantitate the concentration of Polysorbate 80 invaccine formulations. The concentration of detergent is determined bysaponification of the fatty acid moiety; Polysorbate 80 is converted tofree oleic acid by alkaline hydrolysis at 40° C. The sample is separatedby RP-HPLC using a C18 column and quantitated using a UV detector at awavelength of 200 nm.

The first and the second polypeptides are resolved by anion-exchangeHPLC. rLP2086(fHBP) Subfamily A and B proteins elute at distinctretention times and are quantitated using a standard curve generatedagainst the respective rLP2086 protein reference material.

The term “molar ratio” and a description of an immunogenic compositionincluding a fHBP and PS-80 is further disclosed in WO2012025873 and USpatent publication US 2013/0171194, which are each incorporated byreference in their entirety.

The term “molar ratio” as used herein refers to the ratio of the numberof moles of two different elements in a composition. In someembodiments, the molar ratio is the ratio of moles of detergent to molesof polypeptide. In some embodiments, the molar ratio is the ratio ofmoles of PS-80 to moles of protein. In one embodiment, based on theprotein and Polysorbate 80 concentrations, the Molar Ratio may becalculated using the following equation:

${{Molar}\mspace{14mu} {Ratio}} = {\frac{{\% \mspace{14mu} {PS}} - 80}{{mg}\text{/}{ml}\mspace{14mu} {protein}} \times 216}$

In one embodiment, the composition includes about 0.0015, 0.0017,0.0019, 0.0021, 0.0023, 0.0025, 0.0027, 0.0029, 0.0031, 0.0033, 0.0035,0.0037, 0.0039, 0.0041, 0.0043, 0.0045, 0.0047, 0.0049, 0.0051 mg/mLPS-80. Preferably, the composition includes about 0.0035 mg/mL PS-80.

In another embodiment, the composition includes about 10 μg, 11 μg, 12μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21 μg, 22μg, 23 μg, 24 μg, or 25 μg PS-80. In a preferred embodiment, thecomposition includes about 18 μg PS-80.

In another embodiment, the composition includes a PS-80 concentrationranging from 0.0005% to 1%. For example, the PS-80 concentration in thecomposition may be at least 0.0005%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%,0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, or 1.1% PS-80. In a preferred embodiment, thecomposition includes about 0.07% PS-80.

Any minimum value may be combined with any maximum value describedherein to define a range.

Aluminum

The composition preferably includes about 0.5 mg/ml aluminum phosphate.In one embodiment, the composition includes about 0.5 mg aluminum/ml asaluminum phosphate. AlPO₄ at 0.50 mg/ml is added as a stabilizer toprovide enhanced manufacturability and stability. This concentrationmaintains binding (90% binding or better) of the subfamily A and Bproteins to aluminum.

The process for producing an aluminum phosphate is described in USpatent publication US 2009/0016946, which is incorporated by referencein its entirety.

In one embodiment, the composition does not further include amultivalent cation, other than aluminum. In one embodiment, thecomposition does not further include Al(OH)₃ or Al(SO₄)₃.

Excipients

In one embodiment, the composition includes histidine. In oneembodiment, the composition includes sodium chloride. The compositionpreferably includes about 10 mM histidine, and about 150 mM sodiumchloride. In one embodiment, the composition includes 10 mM histidineand 150 mM sodium chloride.

In another embodiment, the composition includes about 650 μg, 660 μg,670 μg, 680 μg, 690 μg, 700 μg, 710 μg, 720 μg, 730 μg, 740 μg, 750 μg,760 μg, 770 μg, 780 μg, 790 μg, 800 μg, 810 μg, 820 μg, 830 μg, 840 μg,or 850 μg of histidine. Preferably, the composition includes about 780μg histidine. Any minimum value may be combined with any maximum valuedescribed herein to define a range.

In one embodiment, the composition includes a tris, phosphate, orsuccinate buffer. In a preferred embodiment, the composition does notinclude tris buffer. In a preferred, the composition does not includephosphate buffer. In one preferred embodiment, the composition does notinclude succinate buffer. In a preferred embodiment, the compositionincludes histidine buffer.

In a preferred embodiment, the pH of the composition is between 6.0 and7.0, most preferably pH 6.0. In one embodiment, the pH of thecomposition is at most 6.1.

Bactericidal Activity

Immune response induced by administering the composition to a human isdetermined using a serum bactericidal assay using human complement(hSBA) against four N. meningitidis serogroup B (MnB) strains. The 4 MnBstrains used in the hSBA were selected from a strain pool. The strainpool represented a collection of systematically collected clinicallyrelevant N. meningitidis serogroup B strains from the US and Europe. Twoof the 4 strains for the SBA are from N. meningitidis serogroup B LP2086(fHBP) subfamily A, and another two of the 4 strains are from N.meningitidis serogroup B LP2086(fHBP) subfamily B.

The high proportion of hSBA response to all test strains, especiallystrains expressing lipoprotein 2086 variants with sequences heterologousto the first polypeptide suggests that the composition is a broadlyprotective vaccine and that two doses are sufficient to confer highseroprotection at least against N. meningitidis serogroup B subfamily Astrains.

The high proportion of hSBA response to all test strains, especiallystrains expressing lipoprotein 2086 variants with sequences heterologousto both the first polypeptide and the second polypeptide suggests thatthe composition is a broadly protective vaccine and that at most threedoses within about a 6 month period are sufficient to confer highseroprotection against N. meningitidis serogroup B strains expressingrLP2086 (FHBP) subfamily A and/or subfamily B.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily Astrain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamilyA strain that expresses a lipoprotein 2086 variant that is heterologousto a N. meningitidis strain expressing A05. For example, in oneembodiment, the hSBA strain is an LP2086 (fHBP) subfamily A strain thatexpresses a lipoprotein 2086 variant that is heterologous to strainM98250771. In one embodiment, the hSBA strain is an LP2086 (fHBP) A22strain. In another embodiment, the hSBA strain is an LP2086 (fHBP) A56strain. In a further embodiment, the hSBA strains are LP2086 (fHBP) A22and LP2086 (fHBP) A56 strains. In another embodiment, the hSBA strain isan LP2086 A04 strain. In one embodiment, the hSBA strain is an LP2086A05 strain. In one embodiment, the hSBA strain is an LP2086 A12 strain.In one embodiment, the hSBA strain is an LP2086 A22 strain. In oneembodiment, the hSBA strain is an LP2086 A12 strain. In one embodiment,the hSBA strain is an LP2086 A04 strain. In one embodiment, the hSBAstrain is an LP2086 A19 strain. In one embodiment, the hSBA strain is anLP2086 A07 strain. In a further embodiment, the hSBA strains includeA22, A12, A19, A05, and A07, or any combination thereof. In oneembodiment, the hSBA strains include A06, A15, and A29, or anycombination thereof.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that is heterologous to a N.meningitidis strain expressing A05. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A22 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA56 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A06 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B A15 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BA29 strain. In one embodiment, the immune response is against N.meningitidis serogroup B A62 strain. In one embodiment, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain that is heterologous to N. meningitidis strain M98250771. Inone embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at least 80%,more preferably at least 84%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the first polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. In a preferredembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily A strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 85%,more preferably at most 99%, identity to a factor H binding proteinexpressed by N. meningitidis strain M98250771. Any minimum value may becombined with any maximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamily Bstrain. In one embodiment, the hSBA strain is an LP2086 (fHBP) subfamilyB strain that expresses a lipoprotein 2086 variant that is heterologousto a N. meningitidis strain expressing B01. For example, in oneembodiment, the hSBA strain is an LP2086 (fHBP) subfamily B strain thatexpresses a lipoprotein 2086 variant that is heterologous to strainCDC1127. In a preferred embodiment, the hSBA strain is an LP2086 (fHBP)subfamily B strain that expresses a lipoprotein 2086 variant that isheterologous to strain CDC1573.

In one embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to a N.meningitidis strain expressing B01. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B24 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB44 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B16 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B03 strain. In oneembodiment, the immune response is against N. meningitidis serogroup BB09 strain. In one embodiment, the immune response is against N.meningitidis serogroup B B15 strain. In one embodiment, the immuneresponse is against N. meningitidis serogroup B B153 strain. In oneembodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that is heterologous to N.meningitidis strain CDC1573. In one embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily B strainthat expresses a factor H binding protein including an amino acidsequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto the second polypeptide. In another embodiment, the immune response isbactericidal against a N. meningitidis serogroup B subfamily B strainthat expresses a factor H binding protein including an amino acidsequence that has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto a factor H binding protein expressed by N. meningitidis strainCDC1573. In a preferred embodiment, the immune response is bactericidalagainst a N. meningitidis serogroup B subfamily B strain that expressesa factor H binding protein including an amino acid sequence that has atleast 80% identity, more preferably at least 87% identity, to a factor Hbinding protein expressed by N. meningitidis strain CDC1573. In anotherpreferred embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has 100% identityto a factor H binding protein expressed by N. meningitidis strainCDC1573.

In another embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to the second polypeptide. Inanother embodiment, the immune response is bactericidal against a N.meningitidis serogroup B subfamily B strain that expresses a factor Hbinding protein including an amino acid sequence that has at most 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to a factor H binding proteinexpressed by N. meningitidis strain CDC1573. In a preferred embodiment,the immune response is bactericidal against a N. meningitidis serogroupB subfamily B strain that expresses a factor H binding protein includingan amino acid sequence that has at most 88% identity, more preferably atleast 99% identity, to a factor H binding protein expressed by N.meningitidis strain CDC1573. Any minimum value may be combined with anymaximum value described herein to define a range.

In one embodiment, the hSBA strain is an LP2086 (fHBP) B24 strain. Inanother embodiment, the hSBA strains is an LP2086 (fHBP) B44 strain. Ina further embodiment, the hSBA strains includes LP2086 (fHBP) B24 andLP2086 (fHBP) B44 strains. In one embodiment, the hSBA strains includesLP2086 (fHBP) A22, LP2086 (fHBP) A56, LP2086 (fHBP) B24, and LP2086(fHBP) B44 strains. In one embodiment, the hSBA strain includes B15. Inone embodiment, the hSBA strain includes B153. In another embodiment,the hSBA strain is an LP2086 B16 strain. In one embodiment, the hSBAstrain is an LP2086 B03 strain. In one embodiment, the hSBA strain is anLP2086 B09 strain. In a further embodiment, the hSBA strains includeB24, B16, B44, B03, and B09, or any combination thereof. In anotherembodiment, the hSBA strains include B24, B16, B44, A22, B03, B09, A12,A19, A05, and A07, or any combination thereof. In another embodiment,the hSBA strains include A06, A07, A12, A15, A19, A29, B03, B09, B15,and B16, or any combination thereof.

In one embodiment, the method induces an immune response against a N.meningitidis serogroup B subfamily A strain and against a N.meningitidis serogroup B subfamily B strain. Preferably, the immuneresponse is bactericidal against a N. meningitidis serogroup B subfamilyA strain and against a N. meningitidis serogroup B subfamily B strain.

In one embodiment, the immune response against the N. meningitidisserogroup B subfamily A strain is greater than the immune responseagainst the N. meningitidis serogroup B subfamily B strain. For example,in one embodiment, the immunogenic composition induces higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain than against a N. meningitidis serogroup B subfamily B strain,when tested under identical conditions. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain occurs within 30 days after a second dose of the immunogeniccomposition against N. meningitidis. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Astrain occur in the absence of a third dose of the immunogeniccomposition against N. meningitidis.

In another embodiment, the immune response against the N. meningitidisserogroup B subfamily B strain is greater than the immune responseagainst the N. meningitidis serogroup B subfamily A strain. For example,in one embodiment, the immunogenic composition induces higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain than against a N. meningitidis serogroup B subfamily A strain,when tested under identical conditions. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain occurs within 30 days after a second dose of the immunogeniccomposition against N. meningitidis. In one embodiment, the higherbactericidal titers against a N. meningitidis serogroup B subfamily Bstrain occur in the absence of a third dose of the immunogeniccomposition against N. meningitidis.

Titers

In one embodiment, the composition induces an increase in bactericidaltiter in the human, as compared to the bactericidal titer in the humanprior to administration of a dose of the composition, when measuredunder identical conditions in an hSBA. In one embodiment, the increasein bactericidal titer is compared to the bactericidal titer in the humanbefore administration of the first dose of the composition, as comparedto the bactericidal titer in the human prior to administration of thefirst dose of the composition, when measured under identical conditionsin an hSBA. In one embodiment, the increase in titer is observed after asecond dose of the composition, as compared to the bactericidal titer inthe human prior to administration of the second dose of the composition,when measured under identical conditions in an hSBA. In anotherembodiment, the increase in bactericidal titer is observed after a thirddose of the composition, as compared to the bactericidal titer in thehuman prior to administration of the third dose of the composition, whenmeasured under identical conditions in an hSBA.

In one embodiment, the composition induces a bactericidal titer in thehuman after administration of a dose, wherein the bactericidal titer isat least greater than 1-fold higher than the bactericidal titer in thehuman prior to administration of the dose, when measured under identicalconditions in an hSBA. For example, the bactericidal titer may be atleast 1.01-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold,14-fold, 15-fold, or 16-fold higher in the human after receiving a doseof the composition, as compared to the bactericidal titer in the humanprior to administration of the dose, when measured under identicalconditions in an hSBA.

In one embodiment, a “responder” refers to a human, wherein thecomposition induces a bactericidal titer in the human afteradministration of a dose, wherein the bactericidal titer is at leastgreater than 1-fold higher than the bactericidal titer in the humanprior to administration of the dose. In a preferred embodiment, theresponder achieves at least a ≧4-fold rise in hSBA titer, as compared toa bactericidal titer in the human prior to administration of the dose.Such a responder may be referred to as having a protective titer.

In one embodiment, the hSBA titer is the reciprocal of the highestdilution of a serum sample that produces a measurable effect. Forexample, in one embodiment, the hSBA titer is the reciprocal of thehighest 2-fold dilution of a test serum that results in at least a 50%reduction of MnB bacteria (50% bacterial survival) compared to the T30CFU value (i.e., the number of bacteria surviving after incubation inassay wells containing all assay components except test serum; 100%bacterial survival).

In one embodiment, the composition induces a bactericidal titer in thehuman after receiving the first dose that is at least 2-fold higher thanthe bactericidal titer in the human prior to receiving the first dose(e.g., higher than the bactericidal titer in the human in the absence ofthe first dose), when measured under identical conditions in the hSBA.In one embodiment, the composition induces a bactericidal titer in thehuman that is at least 4-fold higher than the bactericidal titer in thehuman prior to receiving the first dose, when measured under identicalconditions in a human serum bactericidal assay that utilizes humancomplement (hSBA). In one embodiment, the composition induces abactericidal titer in the human that is at least 8-fold higher than thebactericidal titer in the human prior to receiving the first dose, whenmeasured under identical conditions in a human serum bactericidal assaythat utilizes human complement (hSBA).

In a preferred embodiment, the human serum complement is derived from ahuman having low intrinsic bactericidal activity for a given SBA teststrain. Low intrinsic bactericidal activity refers to, for example, abactericidal titer that is at least less than a 1:4 dilution against thegiven SBA test strain. In one embodiment, the human complement isderived from a human having an hSBA titer that is at least less than1:4, such as a 1:2 dilution, against the given SBA test strain, whereinthe composition was not administered to the human.

A human may exhibit an hSBA titer of less than 1:4 prior toadministration of a composition, such as the bivalent rLP2086composition, or a human may exhibit an hSBA titer of ≧1:4 prior toadministration of the composition. Accordingly, in preferred embodimentsand examples, administration of at least one dose of the composition tothe human results in an hSBA titer that is at least greater than 1:4,such as, for example, an hSBA titer of ≧1.8, an hSBA titer of ≧1:16, andan hSBA titer of ≧1:32. The respective Examples described herein includeassessments of the proportion of human subjects having an hSBA titer≧1:8 and/or ≧1:16, wherein the bivalent rLP2086 composition wasadministered to the human. Such preferred assessments of hSBA titersgreater than 1:4 show that the protection, i.e., the bactericidal immuneresponse induced in the human, is associated with the composition.

In one embodiment, the human has an hSBA titer equal to or greater thanthe hSBA's lower limit of quantitation (LLOQ) after administration ofthe first dose of the composition. In another embodiment, the human hasan hSBA titer equal to or greater than the hSBA's LLOQ afteradministration of the second dose of the composition. In anotherembodiment, the human has an hSBA titer equal to or greater than thehSBA's LLOQ after administration of the third dose of the composition.

Additional Immunogenic Compositions

The inventors surprisingly discovered that the immunogenic compositionagainst N. meningitidis may be administered with an immunogeniccomposition against human papillomavirus (HPV) without negativelyaffecting the bactericidal response against N. meningitidis. Asexplained in Example 7 and Example 8, substantial hSBA responses to N.meningitidis test strains were observed among humans who wereadministered with the immunogenic composition against N. meningitidisand GARDASIL and in humans who were administered with the immunogeniccomposition against N. meningitidis and saline. Additional increases inhSBA responses were observed about 1 month after a third dose of theimmunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immuneresponses against both N. meningitidis and HPV were generated in thehuman following an administration of both the immunogenic compositionagainst N. meningitidis and the immunogenic composition against HPV, ascompared to the immune response in the human before administration ofthe compositions. As explained in Example 7 and Example 8, titersagainst HPV increased in the human after an administration of theimmunogenic composition against N. meningitidis and GARDASIL, ascompared to the titers in the human prior to administration of theimmunogenic compositions. The increase in titers against HPV was atleast greater than 1-fold, at least 2-fold, at least 3-fold, at least4-fold, or more.

Accordingly, in one embodiment, the method includes inducing an immuneresponse against N. meningitidis in a human, wherein the method furtherincludes administering to the human an immunogenic composition againsthuman papillomavirus. Preferably, the immune response is bactericidalagainst N. meningitidis. In one embodiment, the method further includesinducing an immune response against HPV. In a preferred embodiment, themethod further includes inducing an immune response against any one ofhuman papillomavirus types 6, 11, 16, and 18, or any combinationthereof. In one embodiment, the immunogenic composition against HPV isadministered to the human within 24 hours of administering saidcomposition against N. meningitidis.

In one embodiment, the method includes inducing an immune responseagainst N. meningitidis in a human, wherein the method further includesadministering to the human an immunogenic composition against HPV.Preferably, the immune response is bactericidal against N. meningitidis.In one embodiment, the method further includes inducing an immuneresponse against HPV. In a preferred embodiment, the method furtherincludes inducing an immune response against any one of humanpapillomavirus types 6, 11, 16, and 18, or any combination thereof. Inone embodiment, the immunogenic composition against human papillomavirusis administered to the human within 24 hours of administering saidcomposition against N. meningitidis.

In another aspect, the inventors surprisingly discovered that theimmunogenic composition against N. meningitidis may be administered withan immunogenic composition against diphtheria, tetanus, acellularpertussis, and inactivated poliomyelitis virus (dTaP) without negativelyaffecting the bactericidal response against N. meningitidis. Asexplained in Example 4, substantial hSBA responses to N. meningitidistest strains were observed among humans who were administered with theimmunogenic composition against N. meningitidis and REPEVAX. Additionalincreases in hSBA responses were observed about 1 month after a thirddose of the immunogenic composition against N. meningitidis.

Moreover, the inventors surprisingly discovered that robust immuneresponses against both N. meningitidis and dTaP were generated in thehuman following an administration of both the immunogenic compositionagainst N. meningitidis and the immunogenic composition against dTaP, ascompared to the immune response in the human before administration ofthe compositions. As explained in Example 4, titers against dTaPincreased in the human after an administration of the immunogeniccomposition against N. meningitidis and REPEVAX, as compared to thetiters in the human prior to administration of the immunogeniccompositions. The increase in titers against dTaP was at least greaterthan 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, or more.

Methods and Administration

In one aspect, the invention relates to a method of inducing an immuneresponse against N. meningitidis in a human. In another aspect, theinvention relates to a method of vaccinating a human. In one embodiment,the method includes administering to the human at least one dose of thecomposition described above. In another embodiment, the method includesadministering to the human at least a first dose and a second dose ofthe composition described above.

Surprisingly, the inventors discovered that a two-dose schedule of thecomposition induced a bactericidal titer against diverse heterologoussubfamily A and against diverse heterologous subfamily B strains in thehuman. For example, the percentage of humans with an hSBA titer ≧1:8 was90% or greater for SBA test strains expressing LP2086 (fHBP) A22 orLP2086 (fHBP) A56 following a two-dose schedule of the compositiondescribed above. See Example 1.

In one embodiment, the second dose is administered at least 20, 30, 50,60, 100, 120, 160, 170, or 180 days after the first dose, and at most250, 210, 200, or 190 days after the first dose. Any minimum value maybe combined with any maximum value described herein to define a range.

In another embodiment, the second dose is administered about 30 daysafter the first dose. In another embodiment, the second dose isadministered about 60 days after the first dose, such as, for example,in a 0, 2 month immunization schedule. In another embodiment, the seconddose is administered about 180 days after the first dose, such as, forexample, in a 0, 6 month immunization schedule. In yet anotherembodiment, the second dose is administered about 120 days after thefirst dose, such as, for example, in a 2, 6 month immunization schedule.

In one embodiment, the method includes administering to the human twodoses of the composition and at most two doses. In one embodiment, thetwo doses are administered within a period of about 6 months after thefirst dose. In one embodiment, the method does not include furtheradministration of a booster to the human. A “booster” as used hereinrefers to an additional administration of the composition to the human.Administering to the human at most two doses of the composition may beadvantageous. Such advantages include, for example, facilitating a humanto comply with a complete administration schedule and facilitatingcost-effectiveness of the schedule.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 25, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 days, and most 400,390, 380, 370, 365, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260,250, 240, 230, 220, 210, or 200 days after the first dose. Any minimumvalue may be combined with any maximum value described herein to definea range.

In one embodiment, the first dose and the second dose are administeredto the human over a period of about 30 days. In another embodiment, thefirst dose and the second dose are administered to the human over aperiod of about 60 days. In another embodiment, the first dose and thesecond dose are administered to the human over a period of about 180days.

Three Doses

The inventors further surprisingly discovered that a three-dose scheduleof the composition induced a broader bactericidal titer against strainsexpressing heterologous LP2086 (fHBP) subfamily B strains in a greaterpercentage of humans than a two-dose schedule. For example, thepercentage of humans with a hSBA titer ≧1:8 was 65% or greater for SBAtest strains LP2086 (fHBP) B24 and LP2086 (fHBP) B44 following atwo-dose schedule of the composition described above. The percentage ofhumans with a hSBA titer ≧1:8 was 86% or greater for SBA test strainsB24 and B44 following a three-dose schedule of the composition describedabove. See Example 1.

Accordingly, in one embodiment, a three-dose schedule of the compositioninduces a bactericidal titer against multiple strains expressing LP2086(fHBP) heterologous to the first and/or second polypeptide in a greaterpercentage of humans than a two-dose schedule.

In one embodiment, the method includes administering to the human threedoses of the composition. In another embodiment, the method includesadministering at most three doses of the composition. In one embodiment,the three doses are administered within a period of about 6 months afterthe first dose. In one embodiment, the method includes an administrationof a booster dose to the human after the third dose. In anotherembodiment, the method does not include administration of a booster doseto the human after the third dose. In another embodiment, the methoddoes not further include administering a fourth or booster dose of thecomposition to the human. In a further embodiment, at most three doseswithin a period of about 6 months are administered to the human.

In an exemplary embodiment, the second dose is administered about 30days after the first dose, and the third dose is administered about 150days after the second dose, such as, for example, in a 0, 1, 6 monthimmunization schedule. In another exemplary embodiment, the second doseis administered about 60 days after the first dose, and the third doseis administered about 120 days after the second dose, such as, forexample, in a 0, 2, 6 month immunization schedule.

In one embodiment, the first dose, second dose, and third dose areadministered to the human over a period of about 150, 160, 170, or 180days, and at most 240, 210 200, or 190 days. Any minimum value may becombined with any maximum value described herein to define a range.Preferably, the first dose, second dose, and third dose is administeredto the human over a period of about 180 days or 6 months. For example,the second dose may be administered to the human about 60 days after thefirst dose, and the third dose may be administered to the human about120 days after the second dose. Accordingly, an exemplary schedule ofadministration includes administering a dose to the human at aboutmonths 0, 2, and 6.

As described above, multiple doses of the immunogenic composition may beadministered to the human, and the number of days between each dose mayvary. An advantage of the method includes, for example, flexibility fora human to comply with the administration schedules.

EXAMPLES

The following Examples illustrate embodiments of the invention. Unlessnoted otherwise herein, reference is made in the following Examples toan investigational bivalent recombinant vaccine (rLP2086), which is apreferred exemplary embodiment of a composition including 60 μg of afirst lipidated polypeptide including the amino acid sequence set forthin SEQ ID NO: 1 per 0.5 mL dose, 60 μg of a second lipidated polypeptideincluding the amino acid sequence set forth in SEQ ID NO: 2 per 0.5 mLdose, 2.8 molar ratio polysorbate-80 to the first polypeptide, 2.8 molarratio polysorbate-80 to the second polypeptide, 0.5 mg Al³⁺/ml of thecomposition, 10 mM histidine, and 150 mM sodium chloride. Morespecifically, the investigational bivalent recombinant rLP2086 vaccineincludes (a) 60 μg of a first lipidated polypeptide including the aminoacid sequence set forth in SEQ ID NO: 1; (b) 60 μg of a second lipidatedpolypeptide including the amino acid sequence set forth in SEQ ID NO: 2;(c) 18 μg polysorbate-80; (d) 250 μg aluminum; (e) 780 μg histidine, and(f) 4380 μg sodium chloride. Each dose was 0.5 mL.

Example 1 Safety, Tolerability, and Immunogenicity of an InvestigationalMeningococcal Serogroup B Bivalent (MnB) rLP2086 Vaccine in HealthyAdolescents when Administered in Regimens of 2 or 3 Doses in HealthySubjects Aged 11 to 18 Years Background:

Safety, tolerability, and immunogenicity of an investigational bivalent,recombinant vaccine (rLP2086) were studied in healthy adolescents 11-18years of age using 5 dose regimens including 2 or 3 vaccinations (Table1).

The vaccine is a 0.5 ml-dose formulated to contain 60 μg each of apurified subfamily A and a purified subfamily B rLP2086 protein, 2.8molar ratio polysorbate-80, and 0.25 mg of Al³⁺ as AlPO₄, 10 mMhistidine-buffered saline at pH 6.0.

Saline is used as a placebo because there is no known proven safe,immunogenic, and effective vaccine against MnB that could serve as anactive control. The normal saline solution includes 0.9% sodium chloridein a 0.5 ml dose.

Methods:

All subjects in this phase 2, randomized, placebo-controlled,single-blind study attended vaccination visits at months 0, 1, 2 and 6.For blinding, a saline control was given when vaccine was not scheduled.Serum bactericidal assays using human complement (hSBA) were performedwith 4 MnB test strains expressing LP2086 (fHBP) fHBP variants A22, A56,B24 and B44 (i.e., the 4 “primary hSBA test strains” in the primaryendpoint analysis), all of which are different from the variants in thevaccine. Unsolicited adverse events (AE), solicited local and systemicreactions, and antipyretic use were assessed.

Geometric mean hSBA titers were computed for each primary strain at eachblood sampling time point along with 2-sided 95% confidence intervals(CIs). Geometric mean fold rises were computed along with 95% CIs.

A responder was defined as a subject with an hSBA titer equal or abovethe lower limit of quantitation (LLOQ) of the hSBA assays. The LLOQ foreach of the 4 hSBA test strains in the primary endpoint analysis was anhSBA titer equal to 1:8. The limit of detection (LOD) for each primarytest strain was a titer equal to 1:4 (widely viewed as the correlate ofprotection against meningococcal disease).

Results:

1 month after the last vaccine dose, 86-99% subjects (after 3 doses;P<0.001) and 69-100% of subjects (after 2 doses) had hSBA titers ≧8 toeach MnB test strain. After study dose 1, 19-27% (1.1-4.3% severe) and23-27% (0.0-1.0% severe) of rLP2086 recipients experienced redness andswelling, respectively, by group. Injection site pain was the mostcommon local reaction after study dose 1 (7.6-13.1% severe). Fever ≧38°C. after the first study dose of the bivalent rLP2086 vaccine wasexperienced in 3.3-6.5% by group compared to 2.1% in saline recipients.Local and systemic reactions were generally more frequent after dose 1than after subsequent doses. 43 of 1712 subjects (2.5%) reported 51serious AEs; 2 cases were considered related (1 case of vertigo, chillsand headache and 1 case of fever and vomiting). No deaths were reported.

TABLE 1 Table. Statistical Analysis on Proportion of Evaluable StudySubjects Achieving hSBA Titer ≧8* for Each Primary Strain 1 Month AfterLast Dose of Bivalent rLP2086 - Evaluable Immunogenity Population Group1 Group 2 Group 3 (0, 1, 6 mo) (0, 2, 6 mo) (0, 6 mo) Strain [variant]n^(†)/N^(‡) %^(§)(95% CI)^(¶) n^(†)/N^(‡) %^(§)(95% CI)^(¶) n^(†)/N^(‡)%^(§) (95% CI)^(¶) PMB80 [A22] 330/360 91.7^(¶)(88.3, 94.3) 339/35795.0^(¶)(92.1, 97.0) 345/369 93.5^(¶)(90.5, 95.8) PMB2001 [A56] 360/36299.4^(¶)(98.0, 99.9) 355/359 98.9^(¶)(97.2, 99.7) 364/370 98.4^(¶)(96.5,99.4) PMB2948 [B24] 315/354 89.0^(¶)(85.2, 92.0) 313/354 88.4^(¶)(84.6,91.6) 291/359 81.1^(¶)(76.6, 85.0) PMB2707 [B44] 315/356 88.5^(¶)(84.7,91.6) 303/352 86.1^(¶)(82.0, 89.5) 276/356 77.5^(¶)(72.2, 82.3) Group 4Group 5 (0, 2 mo) (2, 6 mo) Strain [variant] n^(†)/N^(‡) % (95% CI)^(¶)n^(†)/N^(‡) % (95% CI)^(¶) PMB80 [A22] 216/238 90.8 (86.3, 94.1) 102/11191.9 (85.2, 96.2) PMB2001 [A56] 240/240 100.0 (98.5, 100.0) 112/113 99.1 (95.2, 100.0) PMB2948 [B24] 173/237 73.0 (66.9, 78.5)  76/110 69.1(59.6, 77.6) PMB2707 [B44] 164/234 70.1 (63.8, 75.9)  81/111 73.0 (63.7,81.0) *Lower limit of quantification for all strains = 8. ^(†)Number ofsubjects with hSBA titer ≧8. ^(‡)Number of subjects with valid hSBAtiters. ^(§)P < 0.001 using one-sided exact test based on binomialdistribution; Values < 0.0125 are considered significant. ^(¶)Exact2-sided confidence interval (Clopper and Pearson) based upon theobserved proportion of subjects.

Conclusions:

Bivalent rLP2086 had an acceptable safety profile. All 5 dosing regimensyielded hSBA titers ≧8 against all 4 test strains in a high proportionof subjects. The higher proportions against some test strains after 3doses compared with 2 doses indicate that 3 doses may provide thebroadest protection against diverse MnB clinical strains. Global phase 3clinical trials are underway with the bivalent rLP2086 vaccine.

One of the objectives of this study was to assess the immune response,as measured by hSBA performed with MnB strains expressing LP2086subfamily A and B proteins, 1 month after the third vaccination withbivalent rLP2086, among Group 1 subjects (0-, 1-, and 6-month scheduleas randomized) and among Group 2 subjects (0-, 2-, and 6-month scheduleas randomized). An endpoint for the immunogenicity analysis was theproportion of subjects in Groups 1 and 2 achieving an hSBA titer≧ LLOQat Month 7 (or 1 month after the third dose of bivalent rLP2086) foreach of the 4 primary MnB test strains (A22, A56, B24, and B44). TheLLOQ was 1:8 for the 4 primary MnB test strains.

For the evaluable immunogenicity population, the proportion of subjectsin Group 1 achieving an hSBA titer ≧1:8 after 3 doses of bivalentrLP2086 was 91.7% for A22, 99.4% for A56, 89% for B24, and 88.5% for B44(See Table 1 above). Since the lower limit of the 97.5% CI was >50% forall strains (87.8%, p<0.001; 97.8%, p<0.001; 84.7%, p<0.001; and 84.1%,p<0.001 for strains A22, A56, B24, and B44, respectively, the studyobjective was met for subjects in Group 1.

For Group 2, the proportion of subjects achieving an hSBA titer ≧1:8after 3 doses of bivalent rLP2086 was 95.0% for A22, 98.9% for A56,88.4% for B24, and 86.1% for B44 (See Table 1 above). Similar to whatwas seen for Group 1, the lower limit of the 97.5% CI was >50% for allstrains (91.7%, p<0.001; 96.9%, p<0.001; 84.1%, p<0.001; and 81.4%,p<0.001 for strains A22, A56, B24, and B44, respectively, demonstratingthat the objective was also met for the subjects in Group 2.

A secondary objective was to assess the immune response, as measured byhSBA performed with MnB strains expressing LP2086 subfamily A and Bproteins, 1 month after the second dose of bivalent rLP2086, among group3 subjects (0- and 6-month schedule as randomized). This secondaryobjective was the proportion of subjects in Group 3 achieving an hSBAtiter ≧LLOQ (1:8) at Month 7 (or 1 month after the second dose ofbivalent rLP2086) for each of the 4 primary MnB test strains.

This secondary objective was also met since the proportion of subjectsin Group 3 achieving an hSBA titer ≧1:8 after 2 doses of bivalentrLP2086 was 93.5%, 98.4%, 81.1%, and 77.5% for the primary MnB teststrains with the lower limit of the 97.5% CI >50% for all strains(90.0%, p<0.001; 96.2%, p<0.001; 76.0%, p<0.001; and 72.2%, p<0.001 forstrains A22, A56, B24, and B44, respectively. See Table 1 above).

Another secondary objective was the proportion of subjects with hSBAtiter LLOQ for each of the 4 primary MnB test strains at each bloodsampling time point for subjects in Groups 1 to 5. The LLOQ for each ofthe 4 primary hSBA test strains was a titer of 1:8. The proportions ofsubjects with an hSBA titer ≧1:8 by study time for the evaluableimmunogenicity population is shown in Table 1 above.

The proportion of subjects who had an hSBA titer ≧1:8 after 1 dose ofbivalent rLP2086 (Group 5 [2- and 6-month schedule] 1 month afterInjection 3) was 55.9% for A22, 67.6% for A56, 56.9% for B24, and 23.8%for B44.

The proportion of subjects who had an hSBA titer ≧1:8 one month after 2doses of bivalent rLP2086 ranged from 74.6% to 100% for subfamily Astrains, and from 54.0% to 81.1% for subfamily B strains. After 3 doses,the proportion increased and ranged from 91.7% to 99.4% and from 86.1%to 89.0% for subfamily A and B strains, respectively.

Example 2 Serum Bactericidal Assay Using Human Complement (HSBA)

MnB clearance from the human bloodstream is primarily achieved bycomplement-mediated bacteriolysis and an intact complement system isimportant for resistance against infections caused by MnB. The in vivocomplement-mediated bacteriolysis of MnB is mimicked in vitro by theserum bactericidal assay using human complement (hSBA), a functionalserological assay shown to be the surrogate of protection formeningococcal disease. That is, demonstration of bacterial killing inthe serum bactericidal assay using human complement (hSBA) correlateswith protection against meningococcal disease. Immunity elicited by thevaccine is determined using hSBAs against 4 MnB strains (fHBP variantsA22, A56, B24, and B44).

The four primary MnB test strains were used in the hSBAs described inthe Examples for the determination of endpoints. That is, these strainswere used to estimate vaccine efficacy using hSBA immunogenicityendpoints. These test strains represent 4 of the 6 fHBP phylogeneticsubgroups that account for >90% of disease isolates circulating in theUSA and Europe.

Identity to matched Lipooligo- fHBP saccharides subfamily Sialationvaccine fHBP Level Variant component subgroup CC PorA (mol %) A56 98.1%N1C2 CC213 P1.22,14 55% B44 91.6% N4/N5 CC269 P1.19- 23% 1,10-4 A2288.9% N2C2 CC41/44 P1.21,16 84% B24 86.2% N6 CC32 P1.12- 22% 1,13-1

In selecting the 4 primary MnB test strains from invasive diseaseisolates, an approach was used which took into account the populationdistribution of the in vitro LP2086 surface expression. Furthermore, thehSBA test strains had to show low baseline hSBA positively, as thepopulations at risk for meningococcal disease are characterized bynon-existing or low baseline bactericidal activity to most strains. Inaddition, each of the 4 primary MnB test strains expresses an LP2086variant that is different from the LP2086 variant in the vaccine, thusallowing an objective assessment of functional immunogenicity andefficacy to invasive meningococcal disease (IMD) strains circulating inthe population.

The hSBA measures the amount of anti-meningococcal serogroup B (MnB)antibody in serum capable of initiating complement-mediated bactericidalactivity. Briefly, test serum is serially-diluted in 2-fold steps andadded to 96-well assay plates. MnB SBA test strains and human serumcomplement are added, initiating the bactericidal reaction. Afterincubation of the assay plates at 37° C. for 30-60 minutes (depending onSBA test strain; called T30), the reaction mixture containing bacteriasurviving this incubation are diluted and transferred to microfilterplates. Following overnight incubation, surviving bacteria expressed ascolony-forming units (CFU) are enumerated using an Immunospot Analyzer.The raw CFU data are recorded electronically and transferred to a dataanalysis application that calculates the hSBA titer. The hSBA titer isthe reciprocal of the highest 2-fold dilution of a test serum thatresults in at least a 50% reduction of MnB bacteria (50% bacterialsurvival) compared to the T30 CFU value (i.e., the number of bacteriasurviving after incubation in assay wells containing all assaycomponents except test serum; 100% bacterial survival). Titers may bereported as step titers, i.e., 1:4, 1:8, 1:16, etc. Serum samples aretested by two individual, replicate determinations in the same assay.The final titer reported for samples in which the replicate measurementsare not identical is the lower of the two replicate measurements whensystem suitability and sample suitability criteria (e.g. replicatetiters must agree within one 2-fold dilution) are met.

hSBA assays were done after serially diluting test sera in Dulbecco'sphosphate-buffered saline. Bacteria (roughly 2000 colony-forming units)and human serum complement (20% by weight final concentration) wereadded to the serially diluted sera in 96-well plates and incubated at37° C. for 30-40 min (depending on hSBA test strain) in a small-radiusorbital shaker at 700 rpm. After incubation, a portion of the reactionmixture was transferred to microfilter plates. After overnightincubation, surviving bacteria were counted with an Immunospot Analyzer(Cellular Technology Limited; Shaker Heights, Ohio, USA) and hSBA titerswere analysed with SAS (version 9.2). The hSBA titer was calculated asthe reciprocal of the interpolated test serum dilution that resulted ina 50% reduction of bacteria compared with a control not subjected totest serum (i.e., surviving bacteria at the end of the hSBA reaction).Per protocol hSBAs were done on the basis of the hSBA titer that was ator above the lower limit of quantitation of the hSBA assays asestablished during qualification of the assays with strains listed inthe Table 1 of Example 1.

Human serum is the complement source for the SBA. However, the hSBAtiters may vary depending on the human complement lot used. Accordingly,human complement is preferably controlled through rigorous screening andqualification to ensure consistent performance in the hSBA. For thehSBA, human serum complement may be pooled from multiple normal healthyhuman adults or used from individual donors (i.e., not pooled).

Example 3 Polysorbate-80

Three parameters have been optimized for drug product formulation: pH,aluminum concentration and polysorbate 80 (PS-80) to protein molarratio. In a dose of the composition having a total volume of 0.5 ml,optimal protein binding to aluminum is achieved at a pH of about 6.0 andabout a 0.5 mg/ml concentration of aluminum as aluminum phosphate(AlPO₄) (which is equivalent to 0.25 mg aluminum per dose). The PS-80 toprotein molar ratio is maintained at 2.8±1.4 in order to stabilize theformulation with respect to in vitro potency. Polysorbate 80 (PS-80) isadded to drug substance to obtain the target PS-80 to protein molarratio of 2.8. Therefore, PS-80 is preferably not added during the drugproduct formulation.

Example 4 Randomized, Placebo-Controlled, Phase 2 Study of theImmunogenicity and Safety of REPEVAX® Administered Concomitantly withBivalent rLP2086 Vaccine in Healthy Adolescents Background/Aims:

The investigational bivalent rLP2086 vaccine, being developed to preventNeisseria meningitidis serogroup B (MnB) disease in adolescents, wasevaluated with concomitant administration of REPEVAX®, adTaP-inactivated polio vaccine (which may be described in U.S. Pat. No.7,479,283, WO1990/013313, and EP1666057 B1, and UK MarketingAuthorisation PL06745/0121) currently used in this population.

Methods:

Adolescents, randomized 1:1 to REPEVAX+rLP2086 or REPEVAX+ saline werevaccinated at 0, 2, and 6 months. The proportion of subjects achievingprespecified antibody levels to 9 REPEVAX antigens 30 days after initialvaccination were determined. Immune responses (hSBA) to 4 MnB teststrains were measured 30 days after vaccinations 2 and 3. Adverse events(AE) and local/systemic reactions were assessed.

REPEVAX (Sanofi Pasteur MSD limited) is a combined low-dose diphtheria,tetanus, acellular pertussis, and inactivated poliomyelitis virusvaccine containing diphtheria toxoid (not less than 2 IU), tetanustoxoid (not less than 20 IU), pertussis antigens (pertussis toxoid (2.5micrograms), filamentous haemagglutinin (5 micrograms), pertacti (3micrograms), and fimbriae Types 2 and 3 (5 micrograms)), polio virus(inactivated) type 1 (40 D antigen units), poliovirus (inactivated type2 (8 D antigen units), poliovirus (inactivated) type 3 (32 D antigenunits), adsorbed on aluminum phosphate (1.5 mg (0.33 mg aluminum)) per0.5-mL dose.

Immune responses to the diphtheria, tetanus, and pertussis components ofREPEVAX (diphtheria toxoid, tetanus toxoid, pertussis toxoid, pertactin,fimbriae types 2 and 3 and filamentous haemagglutinin) were assessedusing a multiplexed LUMINEX assay. Immune responses to poliovirus types1, 2, and 3 were measured in virus neutralization assays. Sera obtainedfrom all subjects in both groups were used in these assays.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with the 4 primary MnB test strainsdescribed. Four primary MnB hSBA test strains (A22, A56, B44, and B24),2 expressing LP2086 subfamily A and the other 2 expressing LP2086subfamily B variants were selected. These 4 primary hSBA test strains(from 4 of the 6 fHBP phylogenetic subgroups and representing >90% ofdisease isolates circulating in the USA and Europe) were used fordetermination of the primary immunogenicity endpoints in this study.Additionally, the A22, B24, and B44 variants are epidemiologicallyrelevant variants in Europe, while in the US, A22 and B24 are the mostprevalent variants found expressed on disease causing MnB strains. TheMnB hSBAs were validated prior to testing of samples used for theprimary and secondary analyses.

Serum samples from 50% of randomly selected subjects in both groups hadhSBA performed with A22 and B24 and the other 50% were tested with A56and B44. These tests were performed on blood samples collected beforeVaccination 1, after Vaccination 2, and after Vaccination 3.

The immunogenicity of REPEVAX is assessed by using prespecified criteriafor each antigen defined in the pivotal Phase 3 clinical trials inadolescents that formed the basis of licensure for REPEVAX. The REPEVAXconcomitant antigens include diphtheria, tetanus, pertussis toxoid,pertussis filamentous hemagglutinin, pertussis pertactin, pertussisfimbrial agglutinogens type 2+3, poliovirus type 1, poliovirus type 2,poliovirus type 3. The exception is for pertussis fimbrial agglutinogens(FIM) types 2+3, which defined a titer of ≧5 EU/mL in the assay used forlicensure of REPEVAX. In this study the lower limit of quantification(LLOQ) of the pertussis FIM types 2+3 assay was ≧10.6 EU/mL, which ishigher and therefore more stringent than the licensing criteria ofREPEVAX.

The LLOQs for the concomitant antigens were 0.037 IU/mL for diphtheriatoxoid; 0.05 IU/ml for tetanus toxoid; 0.9 EU/mL for pertussis toxoid;2.9 EU/mL for pertussis filamentous hemagglutinin, 3.0 EU/mL pertussispertactin; 10.6 EU/mL pertussis fimbrial agglutinogens type 2+3; 1:8 forpoliovirus type 1, poliovirus type 2, poliovirus type 3.

Additional descriptive endpoints for the primary objective were theantibodies to concomitant vaccine antigens measured as geometric meantiter (GMTs) or geometric mean concentrations (GMCs) at postvaccination1 (Visit 2).

Another endpoint was the proportion of subjects with hSBA titer LLOQ atPostvaccination 3 (Visit 6) for each of the 4 primary MnB test strains.

Concomitant Vaccine Antigens.

The proportion of subjects achieving the prespecified criteria for theconcomitant vaccine antigens 1 month after vaccination of diphtheria,tetanus, and pertussis acellular (dTaP)-IPV (REPEVAX) was computed witha 2-sided 95% exact (or Clopper-Pearson confidence limit) for Group 1and Group 2. The difference (bivalent rLP2086/dTaP-IPV-dTaP-IPV, orGroup 1-Group 2) of the proportions was also calculated along with a2-sided 95% exact CI for the difference. Noninferiority was declared ifthe lower limit of the 2-sided 95% CI for the difference was greaterthan −0.10 (−10%) for all of the 9 antigens in the dTaP-IPV vaccine.

hSBAs with Primary Test Strains.

For each primary MnB hSBA test strain, the number and proportion ofsubjects achieving hSBA titers LLOQ, ≧1:4, ≧1:8, ≧1:16, and ≧1:128 ateach blood sampling time point were descriptively summarized along withthe exact 2-sided 95% CI (or Clopper-Pearson confidence limit) for theproportion.

Results:

Of 749 subjects randomized, 685 (91.5%) included the evaluableimmunogenicity population. Immune responses following REPEVAX+rLP2086 orREPEVAX+ saline were noninferior for all 9 REPEVAX antigens. Immuneresponses to the bivalent rLP2086 vaccine were substantial after 2 dosesand further enhanced after 3 doses (Table 2). Mild-to-moderate injectionsite pain was the most common local reaction; headache and fatigue werethe most common systemic events. The proportion of subjects reporting anAE within 30 days postvaccination was similar (8.8% and 11.4%, forREPEVAX+rLP2086 and REPEVAX+ saline, respectively).

For the concomitant vaccine evaluable immunogenicity population, theproportion of subjects achieving the prespecified level of antibodies toconcomitant vaccine antigens (threshold for response) 1 month after theREPEVAX dose was similar between the bivalent rLP2086+REPEVAX group andthe REPEVAX alone group for concomitant vaccine antigens: diphtheriatoxoid (99.4% in each group), tetanus toxoid (100% in each group),pertussis toxoid (94.7% and 96.0%, respectively), pertussis filamentoushemagglutinin (100% in each group), pertussis pertactin (100% in eachgroup), pertussis fimbrial agglutinogens type 2+3 (97.6% and 98.9%,respectively), poliovirus type 1 (100% in each group), poliovirus type 2(100% in each group), poliovirus type 3 (100% in each group).

Noninferiority was achieved because the lower bound of the 2-sided 95%CI for the difference in proportion of responders between the bivalentrLP2086+REPEVAX group (Group 1) and the REPEVAX alone group (Group 2), 1month after the REPEVAX dose was greater than −0.10 (−10%) for the 9antigens in REPEVAX (i.e., the lowest lower bound of the 95% CI on theproportion difference was −4.7% (pertussis toxoid). Hence, the immuneresponse induced by REPEVAX given with bivalent rLP2086 was noninferiorto the immune response induced by REPEVAX alone.

The proportion of subjects with an hSBA titer ≧LLOQ for each of the 4primary MnB test strains for the Postvaccination 3 evaluableimmunogenicity population was assessed. The LLOQ for A22 was an hSBAtiter equal to 1:16 while the LLOQ for all the other MnB test stains wasan hSBA titer equal to 1:8.

For Group 1, the proportion of subjects with an hSBA titer ≧LLOQ atbaseline (before Vaccination 1) was 14.4% for primary MnB strain A22,18.2% for A56, 12.7% for B24, and 6.2% for B44. For Group 2, theproportion of subjects with an hSBA titer ≧LLOQ at baseline (beforeVaccination 1) was 23.0% for primary MnB strain A22, 21.8% for A56,12.9% for B24, and 6.3% for B44.

Substantial hSBA responses were observed among Group 1 subjects afterDose 2 of bivalent rLP2086, with additional increases observed after 3doses 1 month after Vaccination 3. For Group 1 (bivalentrLP2086+REPEVAX), the proportion of subjects achieving an hSBA titer≧LLOQ at 1 month after Vaccination 2 and at 1 month after Vaccination 3was 81.1% and 95.6% for A22, 97.3% and 100% for A56, 81.0% and 96.8% forB24, and 55.5% and 81.5% for B44. While substantial hSBA responses wereachieved after only two bivalent rLP2086 doses, the increase in theproportion of subjects with an hSBA titer ≧LLOQ after 2 doses (1 monthafter Vaccination 2) compared to 3 doses (1 month after Vaccination 3)exemplifies the enhancement of an immune response after 3 doses. In thecontrol group (Group 2), the proportions of subjects with an hSBA titer≧LLOQ for each of the 4 primary MnB test strains at 1 month afterVaccination 2 and 1 month after Vaccination 3 were similar to thebaseline hSBA results for each MnB test strain (before Vaccination 1).

For the 4 primary MnB test strains, the proportion of subjects in Group1 exhibiting a defined hSBA titer was greater after 3 doses than after 2doses. Subjects who achieved an hSBA titer of ≧1:16 are described, sincethis titer is a 4-fold increase from a 1:4 titer (a titer of ≧1:4 iswidely recognized as the correlate of protection against IMD). For Group1, the proportion of subjects with an hSBA titer of 1:16 at 1 monthafter Vaccination 2 was 81.8% for A22, 97.3% for A56, 68.0% for B24, and53.4% for B44. One month after Vaccination 3, the proportion of subjectswith an hSBA titer of 1:16 was 95.6% for A22, 100% for A56, 87.3% forB24, and 79.5% for B44.

In the control group (Group 2), the proportions of subjects exhibitingdefined hSBA titers for each of the 4 primary MnB test strains at 1month after Vaccination 2 and 1 month after Vaccination 3 were similarto the proportion of subjects with the defined hSBA titer at baseline(before Vaccination 1).

For Group 1, the proportion of subjects with an hSBA titer of 1:16following 3 doses of bivalent rLP2086 demonstrated that the vaccineelicits a robust immune response when 3 doses of bivalent rLP2086 wereadministered.

hSBA Geometric Mean Titers (GMTs).

In general, the GMTs at baseline were below the hSBA LLOQs for bothgroups. For Group 1, hSBA GMTs at 1 month after Vaccination 2 were 35.5for A22, 91.1 for A56, 15.9 for B24, and 14.6 for B44. The hSBA GMTs at1 month after Vaccination 3 were 63.4 for A22, 151.5 for A56, 28.3 forB24, and 36.5 for B44.

For Group 1, the observed GMTs after 2 doses for subfamily A strains, aswell as after 3 doses for subfamily B strains, were indicative of arobust immune response.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers for A22, A56, B24, and B44 were assessed. Results fromthe RCDCs in Group 1 showed that substantial immune responses wereobserved among Group 1 subjects after Vaccination 2 of bivalent rLP2086;however, the figures also showed the benefit of a third dose of bivalentrLP2086 as greater proportion of subjects achieved higher titers againstthe 4 MnB test strains. The effect was most pronounced for strain B44.

Conclusions:

When given concomitantly with bivalent rLP2086, REPEVAX induced immuneresponses that were noninferior to those elicited by REPEVAX alone. Thebivalent rLP2086 vaccine induced robust bactericidal responses to fourdiverse MnB test strains, particularly to those representing subfamilyB, that were greater after 3 doses than 2 doses. Concomitantadministration was generally safe and well tolerated.

TABLE 2 Immune response to 4 heterologous MnB test strains after doses 2and 3 of bivalent rLP2086 rLP2086 + REPEVAX Saline + REPEVAX Strain[fHBP variant] HSBA ≧ LLOQ hSBA ≧ LLOQ Time point N^(a) hSBA GMT (95%CI)^(c) n^(b) (%) (95% CI)^(d) N^(a) hSBA GMT (95% CI)^(c) n^(b) (%)(95% CI)^(d) PMB80 [A22] Dose 2 154 35.5 (30.27, 41.61) 126 (81.8)(74.8, 87.6) 166  11.2 (10.02, 12.46) 36 (21.7) (15.7, 28.7) Dose 3 15863.4 (55.29, 72.79) 151 (95.6) (91.1, 98.2) 166 11.0 (9.92, 12.27) 33(19.9) (14.1, 26.8) PMB2001 [A56] Dose 2 149  91.1 (78.00, 106.51) 145(97.3) (93.3, 99.3) 151  8.3 (6.76, 10.29) 39 (25.8) (19.1, 33.6) Dose 3148  151.5 (131.47, 174.59)  148 (100.0)  (97.5, 100.0) 152  8.5 (6.90,10.54) 40 (26.3) (19.5, 34.1) PMB2948 [B24] Dose 2 153 15.9 (13.55,18.55) 124 (81.0) (73.9, 86.9) 167 4.8 (4.41, 5.19) 20 (12.0)  (7.5,17.9) Dose 3 157 28.3 (24.49, 32.66) 152 (96.8) (92.7, 99.0) 170 4.8(4.41, 5.15) 22 (12.9)  (8.3, 18.9) PMB2707 [B44] Dose 2 146 14.6 (11.6,18.43)   81 (55.5) (47.0, 63.7) 159 4.7 (4.24, 5.12) 12 (7.5)   (4.0,12.8) Dose 3 146 36.5 (28.93, 46.18) 119 (81.5) (74.2, 87.4) 159 4.7(4.29, 5.24) 13 (8.2)   (4.4, 13.6) GMT = geometric mean titer; hSBA =serum bactericidal assay using human complement; LLOQ = lower limit ofquantitation (titer 1:16 for PMB80 [A22] and 1:8 for the other MnB teststrains); rLP2086 = recombinant lipoprotein 2086. ^(a)Number of subjectswith valid hSBA titers for the given strain ^(b)Number of subjects withhSBA titer ≧LLOQ for given strain at specified time point ^(c)Confidenceintervals are back transformations of confidence intervals based onStudent t distribution for the mean logarithm of the hSBA titers^(d)Exact 2-sided confidence intervals based on observed proportion ofsubjects using the Clopper and Pearson method

Example 5 Immunogenicity of an Investigational Meningococcal Serogroup BBivalent rLP2086 Vaccine in Healthy Adolescents Background and Aims:

Neisseria meningitidis serogroup B (MnB) causes invasive disease ininfants, adolescents, and adults. A conserved, surface-exposedlipoprotein, LP2086 (a factor H binding protein [fHBP]), is a promisingMnB vaccine target. Safety and immunogenicity of an investigationalbivalent, recombinant vaccine (rLP2086) were studied in healthyadolescents (11-18 years).

Methods:

Subjects in this placebo-controlled, single-blind study were randomizedto two 3-dose schedules and three 2-dose schedules. Each 120-μg dosecontained 2 rLP2086 antigens, 1 from each LP2086 subfamily (A and B).Saline was given when vaccine was not scheduled. Serum bactericidalassays using human complement (hSBA) were performed with 4 MnB teststrains (heterologous to vaccine fHBP).

Results:

1713 subjects (mean age, 14.4 y) were randomized. One month after 3doses of vaccine, hSBA titers ≧8 to subfamily A and B strains wereobserved in 95-99% and 86-89% of subjects, respectively; after 2 doses,these numbers ranged from 91-100% and 69-77% of subjects, respectively.Of the 2-dose schedules, 0 and 6 months induced the highest antibodyresponses (Table 1 of Example 5). hSBA GMTs after 2 doses ranged from6.2-125.6 and after 3 doses ranged from 25.6-155.6 across the 4 MnBheterologous test strains. Mild-to-moderate injection site pain was themost common local reaction. Fever ≧38° C. was experienced in 3.3-6.5%and 2.1% of rLP2086 and saline recipients, respectively, after dose 1.

TABLE 1 Proportion of Subjects Achieving hSBA Titer ≧B

for Each Strain 1 Month After Last Dose of Bivalent rLP2086 Group 1Group 2 Group 3 Group 4 Group 5 (0, 1, 6 mo) (0, 2, 6 mo) (0, 6 mo) (0,2 mo) (2, 6 mo) Strain n = 354-362 n = 352-359 n = 356-370 n = 234-240 n= 110-113 [fHBP variant] % (95% CI)

% (95% CI)

% (95% CI)

% (95% CI)

% (95% CI)

PBM80 [A22] 91.7

 (88.3, 94.3) 95.8

 (82.1, 97.0) 93.6

 (98.5, 95.8) 96.8 (86.3, 94.1) 91.9 (86.2, 96.2) PBM2001 [A56] 98.4

 (98.6, 99.9) 98.9

 (97.2, 99.7) 98.4

 (98.5, 99.4) 100.0 (88.5, 100.0)

(86.2, 100.0) PBM2948 [B24] 89.0

 (85.2, 92.0) 88.4

 (84.6, 91.8) 81.1

 (70.6, 85.0) 73.0 (66.8, 78.5)

(50.0, 77.0) PBM2767 [B44] 88.5

 (84.7, 91.6) 86.1

 (82.9, 88.5) 77.6

 (72.2, 82.3) 70.1 (63.8, 75.9) 73.0 (63.7, 01.0)

= serum bactericidal assay using human complement

Lower limit of quantification for all strong

confidence interval

based upon the observed proportion of subjects.

0.001 values <0.0128

significant p values only apply to Groups 1, 2 and 3.

indicates data missing or illegible when filed

TABLE 2 hSBA GMTs for Each Strain 1 Month After Last Dose of BivalentrLP2086 Group 1 Group 2 Group 3 Group 4 Group 5 (0, 1, 6 mo) (0, 2, 6mo) (0, 6 mo) (0, 2 mo) (2, 6 mo) Strain n = 354-362 n = 352-359 n =356-370 n = 234-240 n = 110-113 [fHBP variant] GMT

 (95% CI)

GMT

 (95% CI)

GMT

 (95% CI)

GMT

 (95% CI)

GMT

 (95% CI)

PBM80 [A22] 66.1 (48.67, 62.67)  56.3 (60.91, 62.27) 48.4 (43.45, 53.86)14.2 (12.08, 16.73) 38.8 (32.31, 48.46) PBM2001  152.9 (137.23, 175.47)155.6 (140.39, 172.38)  125.8 (112.99, 140.17) 28.5 (22.24, 31.58) 111.8(92.73, 134.90) [A56] PBM2948 29.1 (25.88, 22.88) 25.0 (23.08, 28.45) 20.6 (18.38, 23.18) 8.0 (7.01, 0.24)  14.7 (12.01, 18.10) [B24] PBM276740.3 (35.10, 46.11) 35.0 (30.00, 28.81)  22.5 (18.68, 25.72) 6.2 (6.62,7.07)  17.8 (14.12, 22.48) [B44] GMT = geometric mean titer hSBA serumbactericidal assay using human complement

GMTs were calculated using of subjects with 

and determinate hSBA at the given time point

CIs 

back transformation of confidence levels based on the studentsdistribution for the mean logarithm for the hSBA 

indicates data missing or illegible when filed

Conclusions:

rLP2086 was well tolerated. All dosing regimens yielded robustbactericidal responses that were most pronounced after 3 doses.

Table 1 of Example 5 is the same as Table 1 of Example 1, describedabove. Table 2 summarizes the hSBA GMTs and the corresponding CIs bystudy time for the evaluable immunogenicity population. GMTs increasedfrom baseline (before Injection 1) and continued to increase with eachsubsequent dose of bivalent rLP2086.

For the 4 primary MnB strains, the GMTs were greater after 3 doses ofbivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4, and5). The GMTs were similar between the two 3-dose groups, and they weresimilar among the three 2-dose groups.

Before injection 1 (baseline), hSBA GMTs for Groups 1, 2, 3, 4, and 5were as follows: 7.1, 6.3, 6.4, 6.4, and 6.8 for A22, respectively; 6.8,6.1, 6.7, 6.3, and 6.2 for A56, respectively; 5.3, 5.1, 5.0, 4.9, and5.1 for B24, respectively; and 4.4, 4.5, 4.5, 4.6, and 4.4 for B44,respectively.

For Group 1 (0-, 1-, and 6-month), there was a substantial increase inGMTs noted 1 month after Dose 2 for all 4 primary MnB strains (24.4,77.3, 13.8, and 13.1 for A22, A56, B24, and B44, respectively). The GMTsfurther increased after 3 doses of bivalent rLP2086 for Group 1 subjectsfor the 4 primary MnB test strains; 55.1 (A22); 152.96 (A56); 29.1(B24); and 40.3 (B44).

For Group 2, similar increases in GMTs were noted after 2 and 3 doses ofbivalent rLP2086. GMTs for Group 2 subjects after 2 doses of bivalentrLP2086 were 32.9 for A22; 94.6 for A56; 14.9 for B24; and 15.5 for B44.After 3 doses, the GMTs increased to 56.3 for A22; 155.6 for A56; 25.6for B24; and 35.0 for B44.

For Groups 1 and 2, the observed GMTs after 2 doses for subfamily Astrains, as well as after 3 doses for subfamily B strains, areindicative of a robust immune response.

For Group 3, small increases in GMTs were noted after 1 dose of bivalentrLP2086 as follows: 12.0 for A22; 18.5 for A56; 9.2 for B24; and 5.7 forB44. After 2 doses GMTs increased to 48.4 for A22; 125.6 for A56; 20.6for B24; and 22.5 for B44.

For Group 4, GMTs were 13.3 for A22; 17.7 for A56; 9.8 for B24; and 5.9for B44 after 1 dose of bivalent rLP2086. After 2 doses of bivalentrLP2086, GMTs were 37.1 for A22; 104.9 for A56; 17.7 for B24; and 19.1for B44.

For Group 5, GMTs after 1 dose of bivalent rLP2086 were 16.0 for A22;26.8 for A56; 12.6 for B24; and 6.8 for B44. After 2 doses of bivalentrLP2086, the GMTs increased to 39.6 for A22; 111.8 for A56; 14.7 forB24; and 17.8 for B44.

Taken together, for Groups 3, 4, and 5, the observed GMTs are indicativeof an immune response for subfamily A and B strains after 2 doses ofbivalent rLP2086.

In summary, 3 doses of bivalent rLP2086 provided a robust and thebroadest immune response based on the hSBA titers for the 4 primary MnBtest strains. In comparison to 2 doses, a higher proportion of subjectsreceiving 3 doses of bivalent rLP2086 achieved an hSBA titer ≧1:8 to the4 primary MnB test strains.

The results following the 0-, 1-, and 6-month dosing schedule (Group 1)were similar to the results following the 0-, 2-, and 6-month dosingschedule (Group 2). For Groups 1 and 2, the post-Dose 3 GMT valuesachieved were higher than the post-Dose 2 GMT values. For Groups 1 and2, the post-Dose 2 GMT values ranged from 24.4 to 94.6 for subfamily Astrains and from 13.1 to 15.5 for subfamily B strains. The post-Dose 3GMT values ranged from 55.1 to 155.6 for subfamily A strains and from25.6 to 40.3 for subfamily B strains. For Groups 1 and 2, a higherproportion of subjects achieved an hSBA titer ≧1:8 to the 4 primary MnBtest strains following 3 doses of bivalent rLP2086 when compared to theproportion of subjects achieving an hSBA titer ≧1:8 to the 4 primary MnBtest strains after 2 doses of bivalent rLP2086.

Subjects who achieved an hSBA titer of ≧1:16 were also assessed. ForGroup 1, the percentage of subjects who achieved an hSBA titer of ≧1:16one month after 2 doses of bivalent rLP2086 was 73.5% for A22; 96.3 forA56; 57.6 for B24; and 47.2% for B44. Following 3 doses of bivalentrLP2086, the percentage of subjects in Group 1 who achieved an hSBAtiter of ≧1:16 was 91.4% for A22; 99.2% for A56; 82.8% for B24; and84.8% for B44.

For Group 2, the percentage of subjects who achieved an hSBA titer of≧1:16 one month after 2 doses of bivalent rLP2086 was 88.1% for A22;97.9% for A56; 63.5% for B24; and 58.6% for B44. Following 3 doses ofbivalent rLP2086, the percentage of subjects in Group 2 who achieved anhSBA titer of ≧1:16 was 95.0% for A22; 98.9% for A56; 83.6% for B24; and83.8% for B44.

For Groups 1 and 2, the percentage of subjects achieving an hSBA titerof ≧1:16 following 3 doses of bivalent rLP2086 demonstrated that thevaccine elicits a robust immune response.

For Group 3, the percentage of subjects who achieved an hSBA titer of≧1:16 after 2 doses of bivalent rLP2086 was 93.2% for A22; 98.4% forA56; 73.8% for B24; and 70.8% for B44.

For Group 4, the percentage of subjects who achieved an hSBA titer of≧1:16 one month after 2 doses of bivalent rLP2086 was 90.8% for A22;99.2% for A56; 67.1% for B24; and 64.5% for B44.

For Group 5, the percentage of subjects who achieved an hSBA titer of≧1:16 after 2 doses of bivalent rLP2086 was 91.0% for A22; 99.1% forA56; 64.5% for B24; and 66.7% for B44.

For Groups 3, 4, and 5, the percentage of subjects achieving an hSBAtiter of ≧1:16 demonstrated that the vaccine elicits a robust immuneresponse to subfamily A strains following only 2 doses. However, 3 dosesincreases the robustness of response to subfamily B strains.

The percentage of subjects achieving an hSBA titer of ≧1:16 after 3doses of bivalent rLP2086 shows that the vaccine elicits a robust andbroad immune response to MnB strains expressing LP2086 variants that aredifferent from the vaccine components.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers by study times were also assessed for the evaluableimmunogenicity populations for each strain by group. The RCDCs showrobust immune responses after 2 doses of bivalent rLP2086 subfamily Astrains. Following the third dose of bivalent rLP2086, the area underthe response curves increases for all 4 primary MnB test strains,thereby demonstrating the enhancement of the immune response after 3doses of bivalent rLP2086.

The results from the primary and secondary immunogenicity endpointanalyses show that the vaccine can generate antibodies with significanthSBA activity against heterologous subfamily A and subfamily B variantsof MnB. While the proportion of subjects achieving an hSBA titer ≧1:8was higher after 2 or 3 doses of bivalent rLP2086, a large proportion ofsubjects achieved an hSBA titer ≧1:8 one month after 1 dose of bivalentrLP2086. See Group 5 for example.

For the 4 primary MnB test strains, the GMTs were greater after 3 dosesof bivalent rLP2086 (Groups 1 and 2) than after 2 doses (Groups 3, 4,and 5). The GMTs were similar in the two 3-dose groups. The GMTs werealso similar among the three 2-dose groups. These data also demonstraterobust hSBA responses after 3 doses of bivalent rLP2086 based on thepercentages of subjects achieving an hSBA titer ≧1:16.

These data demonstrate that the final formulation of bivalent rLP2086generates a robust immune response and is safe and well tolerated whengiven in 2 or 3 doses. Even 1 dose of bivalent rLP2086 provides asubstantial immune response above baseline and is also safe and welltolerated. Overall, there was no clinically meaningful difference in thesafety profile after 2 or 3 doses of bivalent rLP2086.

Example 6 Safety, Tolerability, and Immunogenicity of a MeningococcalSerogroup B Bivalent rLP2086 Vaccine in Healthy Adolescents Aged 11 to18 Years in Three Phase 2, Randomized, Controlled Studies Background:

Neisseria meningitidis serogroup B (MnB) is a major cause of invasivemeningococcal disease in adolescents. A conserved, surface-exposedlipoprotein, LP2086 (factor H binding protein [fHBP]), is a promisingvaccine target to protect against invasive disease caused by MnB.Safety, tolerability, and immunogenicity of an investigational bivalent,recombinant MnB vaccine (including SEQ ID NO: 1 and SEQ ID NO: 2, 2.8molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mM histidine, and 150mM sodium chloride, herein referred to throughout the Examples as“bivalent rLP2086”) were examined in three phase 2, randomized,controlled studies in healthy adolescents 11-18 years of age.

Methods:

Study 1012 examined 5 vaccine regimens of bivalent rLP2086, whereasstudies 1010 and 1011 evaluated a 3-dose schedule of bivalent rLP2086vaccine given concomitantly with the TdaP-IPV and HPV-vaccines,respectively. Each dose of bivalent rLP2086 contained 60 μg of therLP2086 subfamily A variant A05 and 60 μg of the rLP2086 subfamily Bvariant B01. To examine immunogenicity of bivalent rLP2086 in each ofthe three studies, serum bactericidal assays using human complement(hSBA) were performed with 4 MnB test strains expressing theheterologous fHBP variants A22, A56, B24 and B44, which were selected torepresent relevant diversity of fHBP variability, as well as to providea perspective on the breadth of the vaccine-elicited immune responseagainst strain expressing epidemiologically prevalent fHBP variants.Adverse events and solicited local and systemic reactions were assessed.

Results:

82-100% of subjects in all 3 studies achieved hSBA titers above thelower limit of quantification (LLOQ) for each of the 4 MnB test strains1 month after dose 3 (Table). Across the three studies, the majority ofsystemic events and local reactions were mild to moderate in severity;adverse events were generally not serious or related to the studyvaccine.

Conclusions:

Serum bactericidal antibody titers above 1:4 protect against invasivemeningococcal disease. The demonstration of hSBA titers ≧LLOQ to 4 MnBtest strains, each heterologous to vaccine antigen, in each of theseadolescent phase 2 studies, suggest that the bivalent rLP2086 vaccineprovided a functional antibody response that may be broadly activeagainst diverse MnB disease-associated strains. Vaccinations with thebivalent rLP2086 were generally well tolerated.

TABLE Proportion of Subjects Achieving an hSBA Titer ≧LLOQ for Each fHBPVariant Expressed by Each Test Strain 1 Month After the Last Dose of theBivalent rLP2086 Vaccine fHBP variant expressed % of Subjects by hSBAtest strain A22 A56 B24 B44 Study 1012 (dosing regimen) Group 1 (0, 1, 6mo); n = 354-360 91.4 99.4 89.0 88.5 Group 2 (0, 2, 6 mo); n = 352-35995.0 98.9 88.4 86.1 Group 3 (0, 6 mo); n = 356-370 93.2 98.4 81.1 77.5Group 4 (0, 2 mo); n = 234-240 90.8 100.0 73.0 70.1 Group 5 (0, 4 mo); n= 110-113 91.0 99.1 69.1 73.0 Study 1010 (dosing regimen: 0, 2, 6 mo)rLP2086 + TdaP-IPV Vaccine; n = 146-158 95.6 100.0 96.8 81.5 Study 1011(dosing regimen: 0, 2, 6 mo) rLP2086 + HPV Vaccine; n = 833-849 94.098.9 90.5 82.7 rLP2086 + Saline; n = 847-848 96.3 99.4 92.6 85.7 LLOQ =lower limit of quantification; fHBP = factor H binding protein; hSBA =serum bactericidal assays using human complement; TdaP-IPV Vaccine =Tetanus, Diphtheria, Pertussis, Polio Vaccine. LLOQ = the lowest amountof an analyte in a sample that can be quantitatively determined. hSBAtiters ≧1:4 are a correlate of protection for invasive meningococcaldisease. hSBA titers ≧LLOQ are above the minimal correlate. LLOQ was1:16 for A22; and 1:8 for A56, B24, and B44.

Example 7 Immunogenicity of a Meningococcal Serogroup B Bivalent rLP2086Vaccine in Healthy Adolescents Aged 11 to 18 when AdministeredConcomitantly with Human Papillomavirus Vaccine

This Phase 2, randomized, observer-blind, controlled study evaluated theimmunogenicity of bivalent rLP2086 with or without coadministration withGARDASIL®, which is a quadrivalent vaccine against human papillomavirus(HPV4) (as also described in U.S. Pat. No. 5,820,870), in healthyadolescents ≧11 to <18 years of age. GARDASIL contains recombinantantigens of HPV type 6, 11, 16, and 18 (i.e., HPV-6, HPV-11, HPV-16, andHPV-18) L1 protein. An endpoint was the hSBA GMTs for each of the 4primary MnB test strains at each applicable blood sampling time point.

Methods:

Subjects received bivalent rLP2086 (including SEQ ID NO: 1 and SEQ IDNO: 2, 2.8 molar ratio polysorbate-80, 0.5 mg/ml aluminum, 10 mMhistidine, and 150 mM sodium chloride)+HPV4 (Group 1), bivalent rLP2086+saline (Group 2), or HPV4+ saline (Group 3) at months 0, 2, and 6. Serafrom subjects in Groups 1 and 2 before vaccination 1, and 1 month aftervaccinations 2 and 3, were tested by serum bactericidal assay usinghuman complement (hSBA) using 4 MnB test strains, each expressing anfHBP (A22, A56, B44, and B24) that is heterologous to the vaccinecomponents and represents the breadth of fHBP diversity, as well asepidemiological prevalence. Endpoints assessed included the proportionof subjects with hSBA titers≧ the lower limit of quantitation (LLOQ;1:16 [A22] or 1:8 [A56, B44, B24]) and hSBA geometric mean titers(GMTs).

To demonstrate noninferiority of administrating GARDASIL plus bivalentrLP2086 compared to GARDASIL alone, immunogenicity assessments wereperformed with 2 hSBAs, using 1 primary test strain representingsubfamily A variants (A22) and 1 primary test strain representingsubfamily B variants (B24). However, all 4 primary MnB test strains wereused for determination of additional bivalent rLP2086immunogenicity/efficacy exploratory endpoints.

For assessment of the immune response to bivalent rLP2086, functionalantibodies were analyzed in hSBAs with meningococcal serogroup B strainsrandomly selected from Pfizer's representative MnB SBA strain pool, asdescribed in Example 2. The hSBAs measured the functional antibodies inhuman sera that in a complement-dependent manner kill the targetmeningococcal strain.

Results:

814 and 812 subjects included the evaluable immunogenicity populationfor Groups 1 and 2, respectively. Compared with before vaccination 1,the proportion of subjects with hSBA titers ≧LLOQ against all 4 teststrains was higher after vaccinations 2 (55%-99%) and 3 (83%-99%; FIG.1). Table A of Example 7 presents the hSBA GMTs for each of the 4primary MnB strains and the corresponding CIs by sampling time point forthe evaluable immunogenicity population. The GMTs at baseline were belowthe hSBA LLOQs for both groups. GMTs ranged from 11.1-70.6 and 11.9-76.3after vaccination 1, and 25.8-117.2 and 28.0-128.2 after vaccination 2in Groups 1 and 2, respectively (Table A below).

For the evaluable immunogenicity population, the hSBA GMTs to the 2primary MnB strains at 1 month after the Vaccination 3 bivalent rLP2086dose for Group 1 and Group 2 were as follows: 53.3 and 57.8,respectively for A22 and 25.8 and 28.0, respectively for B24.

For Group 2 (bivalent rLP2086+ saline), hSBA GMTs at 1 month afterVaccination 2 were 33.7 for A22, 76.3 for A56, 16.3 for B24, and 11.9for B44. The hSBA GMTs at 1 month after Vaccination 3 were 57.8 for A22,128.2 for A56, 28.0 for B24, and 31.9 for B44.

For Group 1 (bivalent rLP2086+GARDASIL), hSBA GMTs at 1 month afterVaccination 2 were 31.9 for A22, 70.6 for A56, 15.0 for B24, and 11.1for B44. The hSBA GMTs at 1 month after Vaccination 3 were 53.3 for A22,117.2 for A56, 25.8 for B24, and 27.2 for B44.

Reverse cumulative distribution curves (RCDCs) showing the distributionof hSBA titers for A22, A56, B24, and B44 were assessed for Group 1 andGroup 2 at all sampling time points for the evaluable immunogenicitypopulation. The RCDCs showed that the majority of subjects respondedafter Vaccination 2 and had an additional increase in titer for the 4primary MnB test strains after Vaccination 3. Immune responses to theantigens were similar for Groups 1 and 2.

Conclusions:

Bivalent rLP2086 can be administered with HPV4 without affecting thebactericidal response assessed by hSBA seroresponse or GMTs. Since hSBAtiters ≧1:4 correlate with protection against meningococcal disease,these data indicate the potential for protection of adolescents againsta broad range of MnB strains following administration of the bivalentrLP2086 in the setting of concomitant administration of HPV vaccine.

TABLE A hSBA GMTs - Evaluable Immunogenicity Population Strain [Variant]Group 1 Group 2 Sampling Time rLP2086 + HPV4 rLP2086 + Saline Pointn^(a) GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95% CI)^(c) PMB80 [A22] Before794 9.6 (9.3, 10.0) 799 9.9 (9.5, 10.3) Vaccination 1 1 Month After 79431.9 (29.96, 33.94) 801 33.7 (31.69, 35.85) Vaccination 2 1 Month After803 53.3 (50.22, 56.66) 801 57.8 (54.44, 61.44) Vaccination 3 PMB2001[A56] Before 757 5.0 (4.78, 5.32) 740 5.0 (4.75, 5.28) Vaccination 1 1Month After 790 70.6 (66.17, 75.34) 795 76.3 (71.93, 80.99) Vaccination2 1 Month After 796 117.2 (110.14, 124.76) 802 128.2 (120.65, 136.27)Vaccination 3 PMB2948 [B24] Before 801 4.3 (4.23, 4.46) 793 4.5 (4.35,4.65) Vaccination 1 1 Month After 770 15.0 (13.88, 16.15) 770 16.3(15.15, 17.62) Vaccination 2 1 Month After 788 25.8 (24.14, 27.56) 79328.0 (26.24, 29.87) Vaccination 3 PMB2702 [B44] Before 806 4.1 (4.04,4.15) 805 4.2 (4.10, 4.31) Vaccination 1 1 Month After 783 11.1 (10.21,12.01) 776 11.9 (10.94, 12.96) Vaccination 2 1 Month After 799 27.2(24.99, 29.68) 795 31.9 (29.25, 34.82) Vaccination 3 GMT = geometricmean titer; HPV4 = quadrivalent human papillomavirus vaccine; hSBA =serum bactericidal assay using human complement. ^(a)n = number ofsubjects with valid and determinate hSBA titers for the given strain.^(b)Geometric mean titers were calculated using all subjects with validand determinate hSBA titers at the given time point. ^(c)Confidenceintervals are back transformations of confidence intervals based on theStudent t distribution for the man logarithm of the hSBA titers.

Example 8 Immunogenicity of Human Papilloma Vaccine Coadministered witha Bivalent rLP2086 Vaccine Against Meningococcal Serogroup B in HealthyAdolescents Background:

This Phase 2, randomized study evaluated coadministration of aquadrivalent vaccine against human papillomavirus (HPV4), with bivalentrLP2086, an investigational vaccine against invasive disease caused byNeisseria meningitidis serogroup B (MnB), in healthy adolescents ≧11 to<18 years of age.

Methods:

Subjects received HPV4+ bivalent rLP2086 (Group 1), bivalent rLP2086+saline (Group 2), or saline+HPV4 (Group 3) at months 0, 2, and 6. Serawere collected at baseline and after doses 2 and 3 in all groups. Immuneresponses to HPV4 antigens (HPV-6, 11, 16, and 18) were determined bycompetitive LUMINEX immunoassays (cLIAs). Bivalent rLP2086immunogenicity was measured by serum bactericidal assay using humancomplement (hSBA) with 2 MnB test strains expressingvaccine-heterologous fHBP variants (A22 and B24). Immunogenicityendpoints, all after dose 3, included: geometric mean titers (GMTs)against HPV antigens in Groups 1 and 3; hSBA GMTs for strains expressingvariants A22 and B24 in Groups 1 and 2; and seroconversion rate for HPVantigens in baseline seronegative subjects in Groups 1 and 3. Safety ofbivalent rLP2086 was also assessed after concomitant administration withHPV4 or saline.

Assessments of the immune response to GARDASIL (HPV type 6, 11, 16, and18 L1 protein) were performed using cLIAs based on a fluorescentlylabeled microsphere-based platform (LUMINEX). Sera obtained from allsubjects in Groups 1 and 3 prior to the first vaccination with GARDASIL(Visit 1) and 1 month after the third vaccination with GARDASIL (Visit5) were used in these assays.

The comparison of the GMTs to the 4 HPV antigens for Group 1 and Group3, with their corresponding GMT ratios (GMRs) of Group 1 to Group 3 andthe 2-sided 95% CIs of the ratios is presented in Table A below of thepresent Example. The criterion for the noninferiority margin was1.5-fold, which corresponds to a value of 0.67 for the lower limit ofthe 2-sided 95% CI of the GMR. The 1.5-fold criterion of 0.67 was metfor all the MnB test strains and the HPV antigens except for HPV-18,which had a lower bound 95% confidence interval (CI) of 0.62. In aseparate analysis, ≧99% of subjects seroconverted to all 4 HPV antigensin both the Saline+HPV4 and rLP2086+HPV4 groups.

Another objective of this study was to describe the immune responseinduced by bivalent rLP2086+GARDASIL (Group 1) and by saline+GARDASIL(Group 3), as measured by seroconversion in the HPV immunogenicityassays after the Vaccination 3 dose of GARDASIL (Visit 5) in bothgroups.

The seroconversion rate for each of the 4 HPV antigens, 1 month afterthe last dose of GARDASIL for subjects who were HPV-seronegative atbaseline in Group 1 and Group 3, was calculated as the proportion ofsubjects with anti-HPV serum cLIA levels ≧20 mMU/ml for HPV-6, ≧16mMU/ml for HPV-11, ≧20 mMU/ml for HPV-16, and ≧24 mMU/ml for HPV-18.

The number and proportion of baseline HPV-seronegative subjectsachieving the prespecified criteria for seroconversion for the 4 HPVantigens with the corresponding 95% CIs in each group, the percentdifferences (Group 1-Group 3) in the proportion, and the 95% CIs of thedifferences are presented in Table B of Example 8 for the baselineHPV-seronegative evaluable immunogenicity population.

Results:

The prespecified noninferiority criteria set at 1.5-fold (0.67 lowerlimit of 95% CI for GMRs) were met for 3 of 4 HPV antigens (not HPV-18)and both MnB test strains (Table A). Seroconversion rates in Groups 1and 3 were ≧99% for all HPV antigens (Table B). Greater localreactogenicity occurred after rLP2086 compared with saline but did notincrease with later doses; injection site pain was the most common localreaction. Systemic events in all 3 groups were generally mild andmoderate in severity.

For the evaluable immunogenicity population, the GMTs of antibodies tothe 4 HPV antigens at 1 month after the GARDASIL dose at Vaccination 3for Group 1 and Group 3 were as follows: 451.8 and 550.3, respectively(HPV-6); 892.9 and 1084.3, respectively (HPV-11); 3695.4 and 4763.4,respectively (HPV-16); and 744.0 and 1047.4, respectively (HPV-18). TheGMRs of Group 1 to Group 3 at 1 month after the GARDASIL dose atVaccination 3 were 0.82 for HPV-6 (95% CI: 0.72, 0.94), 0.82 for HPV-11(95% CI: 0.74, 0.91), 0.78 for HPV-16 (95% CI: 0.68, 0.88), and 0.71 forHPV-18 (95% CI: 0.62, 0.81). Therefore, the lower limits of the 2-sided95% CIs for anti-HPV GMRs for Group 1 compared with Group 3 were 0.72for HPV-6, 0.74 for HPV-11, 0.68 for HPV-16, and 0.62 for HPV-18. The1.5-fold criterion of 0.67 (the lower limit of the 2-sided 95% CI of theGMR) was met for all HPV antigens except for HPV-18, which had a lowerbound of the 95% CI of 0.62.

The GMRs of the bivalent rLP2086+GARDASIL group to the bivalent rLP2086+saline group at 1 month after the Vaccination 3 bivalent rLP2086 dosewere 0.92 for A22 (95% CI: 0.85, 1.00), and 0.92 for B24 (95% CI: 0.84,1.01). The lower limits of the 2-sided 95% CIs for the hSBA GMRs forGroup 1 compared with Group 2 were 0.85 for A22 and 0.84 for B24, whichare both greater than 0.67 and therefore met the noninferiority marginof 1.5-fold.

The data from bivalent rLP2086+GARDASIL (Group 1) administration werecompared to data from the bivalent rLP2086+ saline (Group 2)administration by analyzing the hSBA titer 4-fold response rates for 2primary MnB strains (A22 and B24) at 1 month after Vaccination 3 Theproportions of subjects achieving 4-fold rise in hSBA titer frombaseline to 1 month after Vaccination 3 for the 2 primary MnB strainswere measured for both Group 1 subjects who received bivalentrLP2086+GARDASIL and Group 2 subjects who received bivalent rLP2086+saline. Of the subjects in Group 1, 85.3% exhibited ≧4-fold rise in hSBAtiters against B24. Of the subjects in Group 2, 86.4% exhibited ≧4-foldrise in hSBA titers against A22, and 84.8% exhibited ≧4-fold rise inhSBA titers against B24.

The difference in the proportion of responders between Group 1 and Group2 at 1 month after Vaccination 3 was −1.1% for A22 (95% CI: −4.6, 2.3)and −1.4% for B24 (95% CI: −5.1, 2.3). The differences of 4-foldresponse rates were all near a value of 1%, with the lower bounds of the95% CI of the proportion difference being −4.6% A22 and −5.1% B24.

The noninferiority criteria of bivalent rLP2086+GARDASIL compared tosaline+GARDASIL or compared to bivalent rLP2086+ saline required thatthe lower limit of the 2-sided 95% CIs for the GMRs for antibodies toHPV for all 4 HPV antigens (HPV-6, HPV-11, HPV-16, and HPV-18) and forhSBA titers using 2 primary MnB test strains (A22 and B24) 1 month afterVaccination 3 be greater than 0.67. This prespecified criterion was metfor both MnB test strains and at least 3 of the 4 HPV antigens. ForHPV-18, the lower limit of the 2-sided CIs for the GMR was slightlybelow the prespecified threshold of 0.67, at 0.62.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24)were similar (ranged from 83.4% to 86.4%) between the group thatreceived bivalent rLP2086+GARDASIL and the group that received bivalentrLP2086+ saline.

The proportions of subjects in Groups 1 and 2 with prevaccination (i.e.,before Vaccination 1) hSBA titers of ≧1:4 were 15.2% and 18.8%,respectively, for strain A22; 10.4% and 10.5%, respectively, for strainA56; 6.1% and 8.4%, respectively, for strain B24; and 1.7% and 3.2%,respectively for strain B44. In addition, the proportions of subjects inGroups 2 and 1 with prevaccination hSBA titers of ≧1:16 were 13.7% and16.4%, respectively for strain A22; 9.0% and 9.1%, respectively, forstrain A56; 4.1% and 5.4%, respectively, for strain B24; and 1.2% and2.1%, respectively, for strain B44.

In Group 2 (bivalent rLP2086+ saline), the proportion of subjects withan hSBA titer ≧1:4 at 1 month after Vaccination 2 was 86.3% for A22,98.7% for A56, 77.1% for B24, and 60.1% for B44. One month afterVaccination 3, the proportion of subjects with an hSBA titer of ≧1:4 was96.4% for A22, 99.4% for A56, 92.8% for B24, and 86.5% for B44. In Group1 (bivalent rLP2086+GARDASIL), the proportion of subjects with an hSBAtiter of ≧1:4 at 1 month after Vaccination 2 was 83.8% for A22, 97.8%for A56, 71.9% for B24, and 57.7% for B44. One month after Vaccination3, the proportion of subjects with an hSBA titer of ≧1:4 was 94.3% forA22, 99.1% for A56, 91.1% for B24, and 84.4% for B44.

In Group 2 (bivalent rLP2086+ saline), the proportion of subjects withan hSBA titer ≧1:16 at 1 month after Vaccination 2 was 85.8% for A22,98.4% for A56, 68.8% for B24, and 49.9% for B44. One month afterVaccination 3, the proportion of subjects with an hSBA titer of ≧1:16was 96.3% for A22, 99.4% for A56, 89.2% for B24, and 82.4% for B44. InGroup 1 (bivalent rLP2086+GARDASIL), the proportion of subjects with anhSBA titer of ≧1:16 at 1 month after Vaccination 2 was 83.0% for A22,97.2% for A56, 65.2% for B24, and 46.4% for B44. One month afterVaccination 3, the proportion of subjects with an hSBA titer of ≧1:16was 94.0% for A22, 98.9% for A56, 86.3% for B24, and 78.0% for B44.

For both Group 1 and Group 2, a high proportion of subjects achieved anhSBA titer of ≧1:16 or greater following 2 or 3 doses of bivalentrLP2086, while most of the subjects had no measurable hSBA titer to anyof the primary MnB test strains at prevaccination Visit 1.

For the baseline HPV-seronegative evaluable immunogenicity population,the proportion of subjects achieving the prespecified criteria for HPVseroconversion for the HPV antigens at 1 month after the GARDASIL doseat Vaccination 3 for the bivalent rLP2086+GARDASIL group (Group 1) andthe saline+GARDASIL group (Group 3) were as follows: HPV-6 (99.4% and99.3%, respectively), HPV-11 (99.6% and 99.5%, respectively), HPV-16(99.6% and 99.5%, respectively), and HPV-18 (99.5% and 99.0%,respectively).

The difference in proportion of responders between the bivalentrLP2086+GARDASIL group (Group 1) and the saline+GARDASIL group (Group 3)at 1 month after the GARDASIL dose was 0.1% for HPV-6 (95% CI; −0.9,1.5), 0.1% for HPV-11 (95% CI: −0.7, 1.3), 0.1% for HPV-16 (95% CI;−0.7, 1.3), and 0.5% for HPV-18 (95% CI; −0.6, 1.9).

For the bivalent rLP2086+GARDASIL group (Group 1) and thesaline+GARDASIL group (Group 3), the seroconversion rate differenceswere within 0.1% and 0.5% across all 4 HPV antigens and theseroconversion rates were very similar across groups, with greater than99% of subjects seroconverting for all 4 HPV antigens.

As an additional evaluation, bivalent rLP2086+GARDASIL (Group 1) wascompared to bivalent rLP2086+ saline (Group 2), by analyzing the hSBAtiter 4-fold response rates for 2 primary MnB strains (A22 and B24) at 1month after Vaccination 3. The proportions of subjects achieving an hSBAtiter fold rise ≧4 from baseline to 1 month after Vaccination 3 for the2 primary MnB strains are as follows: Of the subjects in Group 1, 85.3%exhibited ≧4-fold rise in hSBA titers against test strain A22, and 83.4%exhibited ≧4-fold rise in hSBA titers against test strain B24. Of thesubjects in Group 2, 86.4% exhibited ≧4-fold rise in hSBA titers againsttest strain A22 and 84.8% exhibited ≧4-fold rise in hSBA titers againsttest strain B24.

The difference in the proportion of responders between Group 1 and Group2 at 1 month after Vaccination 3 was −1.1% for A22 (95% CI: −4.6, 2.3)and −1.4% for B24 (95% CI: −5.1, 2.3). The differences of 4-foldresponse rate were all near a value of 1%, with the lower bounds of the95% CI of the proportion difference being −4.6% (A22) and −5.1% (B24).

Immune Responses to Bivalent rLP2086.

Another objective of this study was to describe the immune response asmeasured by hSBA performed with 4 primary MnB test strains, 2 expressingLP2086 subfamily A proteins (A22 and A56) and 2 expressing LP2086subfamily B proteins (B24 and B44), measured 1 month after the secondvisit (Visit 3) and the third (Visit 5) vaccinations with bivalentrLP2086.

One of the endpoints for this objective was the proportion of subjectswith hSBA titers ≧LLOQ at 1 month after Vaccination 2 (Visit 3) and at 1month after Vaccination 3 (Visit 5) for each of the 4 primary MnB teststrains. The proportion of subjects with hSBA titer ≧LLOQ for each ofthe 4 primary MnB test strains for the evaluable immunogenicitypopulation was assessed. The LLOQ for A22 was an hSBA titer equal to1:16, while the LLOQ for all the other MnB test strains was an hSBAtiter equal to 1:8.

For Group 2 (bivalent rLP2086+ saline), the proportion of subjects withan hSBA titer ≧LLOQ at baseline (before Vaccination 1) was 16.4% forA22, 9.3% for A56, 6.9% for B24, and 2.5% for B44. For Group 2, theproportions of subjects achieving an hSBA titer ≧LLOQ at 1 month afterVaccination 2 and at 1 month after Vaccination 3 were 85.8% and 96.3%,respectively, for A22; 98.5% and 99.4%, respectively, for A56; 74.2% and92.6%, respectively for B24; and 57.1% and 85.7%, respectively, for B44.

For Group 1 (bivalent rLP2086+GARDASIL), the proportion of subjects withan hSBA titer ≧LLOQ at baseline (before Vaccination 1) was 13.7% forA22, 9.2% for A56, 5.1% for B24, and 1.4% for B44. For Group 1, theproportions of subjects achieving an hSBA titer ≧LLOQ at 1 month afterVaccination 2 and at 1 month after Vaccination 3 were 83.0% and 94.0%,respectively, for A22; 97.5% and 98.9%, respectively, for A56; 70.6% and90.5%, respectively for B24; and 54.5% and 82.7%, respectively, for B44.

Substantial hSBA responses to the 4 primary MnB test strains wereobserved among both Group 1 and Group 2 subjects at 1 month afterVaccination 2, with additional increases observed at 1 month afterVaccination 3.

The proportion of subjects achieving an hSBA titer fold rise ≧4 for eachof the 4 primary MnB test strains and the proportions of subjectsachieving the composite response for the evaluable immunogenicitypopulation were assessed. The proportions of subjects with an observedhSBA titer ≧LLOQ for all 4 MnB strains combined at baseline (beforeVaccination 1) were similar between Group 1 (0.3%) and Group 2 (0.7%).

For Group 2 (bivalent rLP2086+ saline), the proportion of subjectsachieving an hSBA titer fold rise ≧4 from baseline to 1 month afterVaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7%for B44, and 83.9% of subjects achieved a composite hSBA response(hSBA≧LLOQ for all 4 primary strains combined). At 1 month afterVaccination 2, the proportion of subjects achieving an hSBA titer foldrise ≧4 from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24,and 47.4% for B44, and 51.9% of subjects achieved a composite hSBAresponse.

For Group 1 (bivalent rLP2086+ saline), the proportion of subjectsachieving an hSBA titer fold rise ≧4 from baseline to 1 month afterVaccination 3 was 86.4% for A22, 95.3% for A56, 84.8% for B24, and 80.7%for B44, and 83.9% of subjects achieved a composite hSBA response(hSBA≧LLOQ for all 4 primary strains combined). At 1 month afterVaccination 2, the proportion of subjects achieving an hSBA titer foldrise ≧4 from baseline was 74.2% for A22, 92.6% for A56, 63.4% for B24,and 47.4% for B44, and 51.9% of subjects achieved a composite hSBAresponse.

Additional hSBA Fold Response.

Other endpoints were the proportion of subjects achieving at least2-fold and 3-fold hSBA titer increases from baseline to eachpostvaccination blood sampling visit for each of the 4 primary MnBstrains. Note that the LLOQ for A22 was an hSBA titer equal to 1:16,while the LLOQ for all the other MnB test strains was an hSBA titerequal to 1:8.

The proportion of subjects achieving a ≧2-fold rise in hSBA titer frombaseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnBstrains were 77.3% and 81.1%, respectively, for A22; 94.4% and 95.3%,respectively, for A56; 63.0% and 66.0%, respectively, for B24; and 46.1%and 48.6%, respectively, for B44. The proportions of subjects achievingan hSBA titer fold rise ≧2 from baseline to 1 month after Vaccination 3for Group 1 and Group 2 for MnB strains were 90.2% and 92.8%,respectively, for A22; 97.2% and 97.9%, respectively, for A56; 84.6% and87.2%, respectively, for B24; and 77.7% and 81.7%, respectively, forB44.

The proportions of subjects achieving an hSBA titer fold rise ≧3 frombaseline to 1 month after Vaccination 2 for Group 1 and Group 2 for MnBstrains were 73.1% and 74.2%, respectively, for A22; 92.5% and 92.6%,respectively, for A56; 61.3% and 63.4%, respectively, for B24; and 45.7%and 47.4%, respectively, for B44. The proportions of subjects achievingan hSBA titer fold rise ≧3 from baseline to 1 month after Vaccination 3for Group 1 and Group 2 for MnB strains were 85.3% and 86.4%,respectively, for A22; 95.0% and 95.3%, respectively, for A56; 83.4% and84.8%, respectively, for B24; and 77.0% and 80.7%, respectively, forB44.

In summary of the descriptive endpoints under the objectives, themajority of subjects achieved an hSBA titer ≧LLOQ for both Group 1(bivalent rLP2086+GARDASIL) and group 2 (bivalent rLP2086+ saline) forall 4 primary MnB test strains, while only a very small proportion ofsubjects had measurable hSBA titers ≧LLOQ at baseline (prevaccinationVisit 1). Substantial immune responses with the 4 MnB strains wereobserved at 1 month after Vaccination 2, with additional increasesobserved at 1 month after Vaccination 3 for both Group 1 and Group 2subjects. This conclusion was confirmed by the proportion of subjectswith an hSBA titer of ≧1:16 following 3 doses, the observed GMTsachieved after 2 doses and after 3 doses in both groups, and the RCDCsfor the 4 primary MnB test strains.

For both Group 1 and Group 2, a high proportion of subjects achieved anhSBA titer fold rise ≧4 for each of the primary MnB test strains and acomposite hSBA response≧LLOQ for all 4 primary MnB strains after thethird study vaccination.

In addition, the majority of subjects achieved an hSBA titer fold rise≧3 and an hSBA titer fold rise ≧2 for the 4 primary MnB strains at allsampling time points for both Group 1 (bivalent rLP2086+GARDASIL) andGroup 2 (bivalent rLP2086+ saline). The proportion of subjects withresults meeting these criteria was higher after 3 vaccinations comparedwith 2 vaccinations.

These results support the evidence that the immune response to bivalentrLP2086 when coadministered with the HPV vaccine, GARDASIL, yields arobust immune response that is comparable to the immune response tobivalent rLP2086+ saline.

HPV GMTs. Table B of Example 8 presents the GMTs and the correspondingCIs for each of the 4 HPV antigens at 1 month after Vaccination 3 forGroup 1 (bivalent rLP2086+GARDASIL) and Group 3 (saline+GARDASIL) in theevaluable immunogenicity population.

For Group 3, the HPV GMTs at baseline (before Vaccination 1) and at 1month after Vaccination 3 were 6.0 and 550.3, respectively, for HPV-6;4.3 and 1084.3, respectively, for HPV-11; 6.1 and 4763.4, respectively,for HPV-16; and 5.3 and 1047.4, respectively, for HPV-18. For Group 1(bivalent rLP2086+GARDASIL), the HPV GMTs at baseline (beforeVaccination 1) and at 1 month after Vaccination 3 were 5.8 and 451.8,respectively for HPV-6; 4.2 and 892.9, respectively, for HPV-11; 5.8 and3695.4, respectively, for HPV-16; and 5.2 and 744.0, respectively, forHPV-18. Overall, the GMTs were numerically higher for Group 3 comparedwith Group 1. Reverse cumulative distribution curves (RCDCs) showing thedistribution of titers for HPV-6, HPV-11, HPV-16, and HPV-18 wereassessed for Group 1 (bivalent rLP2086+GARDASIL) and Group 3(saline+GARDASIL) at all sampling time points for the evaluableimmunogenicity population. The RCDCs showed robust immune responsesamong subjects after Vaccination 3 for both Group 1 and Group 3.

Summary of Immune Response to GARDASIL.

The GMTs to HPV antigens were numerically higher for Group 3(saline+GARDASIL) as compared with Group 1 (bivalent rLP2086+GARDASIL),and the observed HPV GMTs after Vaccination 3 were indicative of arobust immune response for both groups. RCDCs also supported robustimmune responses after Vaccination 3 for both Group 1 and Group 3. Thiswas also supported by the proportion of subjects with seropositivestatus for the 4 HPV antigens, which was >99% at 1 month afterVaccination 3 for both groups. The younger age subgroup had higher HPVGMTs in Group 3 (saline+GARDASIL) than the older age subgroup. Thisdifference was maintained when GARDASIL was given concomitantly withbivalent rLP2086.

Immunogenicity Conclusions.

The noninferiority criteria of bivalent rLP2086_GARDASIL compared tosaline+GARDASIL or compared to bivalent rLP2086+ saline required thatthe lower limit of the 2-sided 95% CIs for the geometric mean titerratios (GMRs) for antibodies to HPV for all 4 HPV antigens (HPV-6,HPV-11, HPV-16, and HPV-18) and for hSBA titers using 2 primary MnB teststrains (A22 and B24) 1 month after Vaccination 3 be greater than 0.67.This prespecified threshold was met for both MnB strains and 3 of the 4HPV antigens. For HPV-18, the lower limit of the 2-sided 95% CIs for theGMR was slightly below the prespecified threshold of 0.67, at 0.62.

Seroconversion for all 4 HPV antigens was achieved by 99% or more of thesubjects for the groups that received GARDASIL concomitantly withbivalent rLP2086 or with saline. The RCDCs for all 4 HPV antigens showthat the majority of subjects achieved a response above theseroconversion threshold at 1 month after Vaccination 3. Robust GMTsrelative to baseline were observed for both groups that receivedGARDASIL.

The 4-fold rise responses to 2 primary MnB test strains (A22 and B24)were similar (ranged from 83.4% to 86.4%) between the group thatreceived bivalent rLP2086+GARDASIL (85.3% and 83.4%, respectively) andthe group that received bivalent rLP2086+ saline (86.4% and 84.8%,respectively).

Further descriptive analyses of the response to bivalent rLP2086 wereperformed using 4 primary MnB test strains (A22, A56, B24, and B44). Ahigh proportion of subjects achieved an hSBA titer fold rise ≧4 and thecomposite response (all 4 primary MnB test strains and the sameimmunogenicity/efficacy endpoint definition as used in the Phase 3clinical program) for the evaluable immunogenicity population for bothgroups that received bivalent rLP2086, either concomitantly withGARDASIL (bivalent rLP2086+GARDASIL) or with saline (bivalent rLP2086+saline), 1 month after Vaccination 2 or 3. These responses aresubstantially higher than an hSBA titer ≧1:4 that has been demonstratedto correlate with protection against meningococcal disease includingserogroup B disease. These results also indicate and support theevidence of a robust immune response to bivalent rLP2086 whetheradministered with saline or concomitantly with GARDASIL.

Conclusions:

Data indicate that robust immune responses to both vaccines weregenerated after concomitant administration of rLP2086+HPV4. Prespecifiednoninferiority criteria were met for 5 of 6 antigens. Although GMRs toHPV-18 narrowly missed noninferiority criteria, the high proportion ofresponders (≧99%) indicates clinical effectiveness is expected to bemaintained after concomitant administration. Bivalent rLP2086 was welltolerated and elicited a robust immune response to test strainsexpressing fHBPs heterologous to those in the vaccine.

TABLE A Comparison of Geometric Mean Titers at 1 Month After Vaccination3 (Evaluable Immunogenicity Population) Group 1 Group 2 Group 3 StrainrLP2086 + HPV4 rLP2086 + Saline Saline + HPV4 Ratio^(d) [Variant] n^(a)GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95% CI)^(c) n^(a) GMT^(b) (95%CI)^(c) (95% CI)^(e) HPV antigens (Group 1 vs Group 3) HPV-6 813 451.8(417.5, 489.0) NA 423  550.3 (490.4, 617.6)  0.82 (0.72, 0.94) HPV-11813 892.9 (839.5, 949.6) 423 1084.3 (997.3, 1179.0)  0.82 (0.74, 0.91)HPV-16 813  3695.4 (3426.3, 3985.7) 423 4763.4 (4285.9, 5294.2) 0.78(0.68, 0.88) HPV-18 813 744.0 (687.7, 805.0) 423 1047.4 (939.0, 1168.3) 0.71 (0.62, 0.81) hSBA strains (Group 1 vs Group 2) PMB80 [A22] 803 53.3(50.2, 56.7)  801 57.8 (54.4, 61.4) NA 0.92 (0.85, 1.00) PMB2948 [B24]788 25.8 (24.1, 27.6)  793 28.0 (26.2, 29.9) 0.92 (0.84, 1.01) CI =confidence interval; GMT = geometric mean titer; HPV = humanpapillomavirus; hSBA = serum bactericidal assay using human complement;LLOQ = lower limit of quantitation; NA = not applicable. Note: LLOQ = 11mMU/ml for HPV-6, 8 mMU/ml for HPV-11; 11 mMU/ml for HPV-16; and 10mMU/ml for HPV-18. LLOQ = 1:16 for A22; 1:8 for A56, B24, and B44.Results below the LLOQ were set to 0.5 * LLOQ for analysis. ^(a)n =number of subjects with valid and determinate assay results for thegiven antigen or strain. ^(b)Geometric mean titers (GMTs) werecalculated using all subjects with valid and determinate assay resultsat 1 month after Vaccination 3. ^(c)Confidence intervals (CIs) are backtransformations of confidence levels based on the Student t distributionfor the mean logarithm of assay results. ^(d)Ratios of GMTs (Group1/Group 3 for HPV antigen titers and Group 1/Group 2 for hSBA straintiters). ^(e)Confidence Intervals (CIs) for the ratio are backtransformations of a confidence interval based on the Student tdistribution for the mean difference of the logarithms of themeasures(Group 1 - Group 3 for HPV titers and Group 1 - Group 2 for hSBAstrain titers).

TABLE B Comparison of Subjects Achieving HPV Seroconversion at 1 MonthAfter Vaccination 3 - Baseline HPV Seronegative Evaluable ImmunogenicityPopulation Group 1 Group 3 Seropositive rLP2086 + HPV4 Saline + HPV4Difference Antigen Criteria N^(a) n^(b) (%) (95% CI)^(c) N^(a) n^(b) (%)(95% CI)^(c) (%)^(d) (95% CI)^(e) HPV-6 ≧20 mMU/mL 802 797 (99.4) (98.6,99.8) 414 411 (99.3) (97.9, 99.9) 0.1 (−0.9, 1.5) HPV-11 ≧16 mMU/mL 801798 (99.6) (98.9, 99.9) 417 415 (99.5) (98.3, 99.9) 0.1 (−0.7, 1.3)HPV-16 ≧20 mMU/mL 800 797 (99.6) (98.9, 99.9) 413 411 (99.5) (98.3,99.9) 0.1 (−0.7, 1.3) HPV-18 ≧24 mMU/mL 805 801 (99.5) (98.7, 99.9) 418414 (99.0) (97.6, 99.7) 0.5 (−0.6, 1.9) CI = Confidence interval; HPV =human papillomavirus. ^(a)N = number of subjects with baseline HPVseronegative status for the given antigen. ^(b)n = Number of subjectsachieving seroconversion (prespecified criteria) at 1 month afterVaccination 3 for the given antigen. ^(c)Exact 2-sided confidenceinterval (Clopper and Pearson) based upon the observed proportion ofsubjects. ^(d)Difference in proportions, expressed as a percentage.^(e)Exact 2-sided confidence interval (based on Chan & Zhang) for thedifference in proportions, expressed as a percentage.

Example 9 Bivalent RLP2086 Vaccine Efficacy

The efficacy of bivalent rLP2086 has been inferred using hSBA responsesas the surrogate of efficacy and demonstration of serum bactericidalantibody responses to invasive N. meningitidis serogroup B (MnB)strains.

Four MnB strains, representative of invasive meningococcal disease (IMD)causing strains, were used in the evaluation. Each MnB test strainexpresses an fHBP protein variant (A22, A56, B24 or B44) that isheterologous (differs) from the vaccine components (A05 and B01).

The efficacy of bivalent rLP2086 was assessed in 3 randomized controlledPhase II studies conducted in 4,459 adolescents aged 11 through 18 yearsof age in the US and Europe. See also Example 6. A total of 2,293received at least 1 dose of 120 μg of bivalent rLP2086 using a 0-, 2-,and 6-month vaccination schedule. Efficacy was assessed by evaluatinghSBA immune responses in subjects vaccinated with bivalent rLP2086.

Efficacy was inferred using 5 co-primary immunogenicity endpoints. For 4of the 5 co-primary endpoints, pre-specified proportions of subjects hadto achieve 4-fold rises in hSBA titer to each of the 4 MnB test strainsfollowing 3 doses of bivalent rLP2086. The fifth co-primary endpoint wasa composite endpoint requiring that a prespecified high proportion ofsubjects each respond in all 4 hSBAs with the primary MnB test strainsfollowing 3 doses of bivalent rLP2086. Immune response was also assessedbased on the proportion of subjects who achieved an hSBA titer≧ thelower limit of quantitation (LLOQ) 1 month after the third dose ofvaccine. LLOQ is defined as the lowest amount of the antibody in asample that can be measured.

Study 1 (described in Example 7 and Example 8) was a Phase II,randomized, active-controlled, observer-blinded, multicenter trial inwhich 2,499 US subjects, 11 through 17 years of age, were randomlyassigned (in a 2:2:1 ratio) to 1 of 3 groups: Group 1 received bivalentrLP2086+HPV4, Group 2 received bivalent rLP2086+ Saline, and Group 3received Saline+HPV4. All vaccinations were administered on a 0-, 2-,and 6-month schedule.

Study 2 (described in Example 4) was a Phase II, randomized,placebo-controlled, single-blind trial in which 753 European subjects,11 through 18 years of age, were randomly assigned in a 1:1 ratio to 2groups:Group 1 received bivalent rLP2086 at 0-, 2-, and 6-months anddTaP-IPV (diphtheria, tetanus, acellular pertussis-inactivated poliovirus) at Month 0. Group 2 received Saline at 0-, 2-, and 6-months anddTaP-IPV at Month 0.

Study 3 (described in Example 5) was a Phase II, randomized,placebo-controlled, single-blind, multicenter trial in which 1,713European subjects, 11 through 18 years of age, were randomly assigned ina 3:3:3:2:1 ratio to 5 groups. Subjects received 2 or 3 doses ofbivalent rLP2086 administered on a 0-, 1-, and 6-month schedule (Group1); on a 0-, 2-, and 6-month schedule (Group 2); on a 0- and 6-monthschedule (Group 3); on a 0- and 2-month schedule (Group 4); or on a 0-and 4-month schedule (Group 5). Saline injections (1 or 2 dosesdepending on group) were administered in each group to maintain theblind.

Results in Studies 1, 2, and 3 among subjects who received a 3-doseseries of bivalent rLP2086 at 0-, 2-, and 6-months are described abovein the respective Examples 4-8. Evaluation of the 4-fold and compositeresponse rates were exploratory endpoints for all studies. The 4-foldresponse rates showed that the lower bounds of the 95% ConfidenceInterval (CI) for all 4 endpoints were similar among the 3 studies andconsistently met the threshold limits for the Phase III endpoints. Theproportion of subjects achieving hSBA titer≧LLOQ was similar across the3 studies.

Based on the hSBA data acquired following 2 administrations of thevaccine given 1 or 2 months apart, 2 doses of vaccine administered overthese intervals may provide protection to individuals at increased risk,due to potential exposure to a case of meningococcal serogroup Bdisease. The responses observed after 2 vaccine administrationsdelivered 1 or 2 months apart showed that a proportion of subjectsexpressed hSBA levels equal to or above the LLOQ values for each of the4 primary test strains (see Study 1 results for Group 1 and Group 2; seeStudy 2 results for Group 1; see Study 3 results for Group 2). A thirddose of the vaccine, administered at 6 months, can achievevaccine-mediated protection.

Concomitant Vaccine Administration.

Study 1 (described in Example 7 and Example 8) evaluated the concomitantuse of bivalent rLP2086 and HPV4 in US adolescents. The study endpointsincluded noninferiority assessment of the immune response for the fourHPV4 antigens (based on geometric mean titer [GMT]) and for bivalentrLP2086 (based on hSBA using two MnB test strains [variants A22 andB24]) 1 month after the third vaccination. HPV4 immune response was alsoevaluated by seroconversion for each of the 4 HPV antigens.

Study 1 shows the comparison of the geometric mean titers (GMTs) of theantibodies to HPV antigens for Group 1 (bivalent rLP2086+HPV4) and Group3 (Saline+HPV4), with their corresponding GMT ratio (GMRs) between Group1 and Group 3 and the 2-sided 95% CIs of the ratios. Study 1 alsoprovides the comparison of hSBA GMTs to the 2 primary MnB test strainsfor Group 1 and Group 2 with their corresponding GMRs between Group 1and Group 2 and the 2-sided 95% CI of the ratios. The criterion fornoninferiority margin was 1.5-fold, which corresponds to a value of 0.67for the lower limit of the 2-sided 95% CI of the GMR. The 1.5-foldcriterion of 0.67 was met for all the MnB test strains and the HPVantigens except for HPV-18, which had a lower bound 95% confidenceinterval (CI) of 0.62. Although the response to HPV-18 did not meet thepre-specified noninferiority criterion, the difference was marginal. Ina separate analysis, ≧99% of subjects seroconverted to all 4 HPVantigens in both the Saline+HPV4 and bivalent rLP2086+HPV4 groups.

Example 10 Bivalent rLP2086 Elicits Antibodies in Individuals thatProvide Broad Coverage Against MnB Strains Expressing Prevalent andOutbreak-Associated fHBP Variants

Bactericidal antibodies measured in serum bactericidal assays usinghuman complement (hSBAs) have been correlated with protection frommeningococcal disease and hSBA responses have been used routinely assurrogates of vaccine efficacy. Global epidemiological studies of fHBPdiversity revealed that ˜80% of meningococcal disease is caused bystrains that express one of 10 prevalent fHBP variants.

Methods:

hSBA responses to Neisseria meningitidis serogroup B (MnB) strainsexpressing the 10 most prevalent fHBP variants in the US and Europe(B24, B16, B44, A22, B03, B09, A12, A19, A05 and A07) in individualhuman subjects immunized with bivalent rLP2086 were evaluated. MnBstrains expressing these ten most prevalent variants represent thebreadth of fHBP diversity, including 5 of the 6 major fHBP subgroups,that are representative of >98% and 97% of strains (by subgroup) in theMnB SBA strain pool, and US subset of the MnB SBA strain pool,respectively. Twenty-three MnB test strains were obtained from Pfizer'sMnB SBA strain pool (N=1263) that represent strains systematicallycollected from the US and Europe between the years 2000 and 2006. Inaddition, isolates from recent MnB disease outbreaks were included inthe analysis. Matched prevaccination and postvaccination sera (postdose2 and postdose 3) were obtained randomly from adolescent and young adultsubjects enrolled in clinical studies B1971005, B1971012 or B1971003.

To provide additional information supporting the potential coverageafforded by vaccination with bivalent rLP2086, hSBAs were performed withthe outbreak strains and serum samples from nine subjects immunized withbivalent rLP2086 (clinical study B1971012, described in Example 5 andExample 6. The subjects (11 to <19 years of age) had received 3 doses ofbivalent rLP2086 at 0, 2 and 6 months. To ensure a conservative hSBAassessment the nine subjects were selected in a non-biased manner from aset of subjects with no baseline hSBA activity against the primary MnBtest strains. Two of the clonal Princeton University outbreak strains(PMB5021 and PMB5025) and two of the UCSB outbreak strains (one fromeach of the two genetic clusters, PMB4478 and PMB4479, were tested.

Genetic characterization of the clonal Princeton University MnB OutbreakStrains is as follows: data suggest that the Princeton Universityoutbreak strains are clonal. Each of the strains was typed as CC41/44(ST 409) and expressed fHBP variant B153 (SEQ ID NO: 6). The strains hadidentical allele assignments for NHBA (2), porA (subtype P1.5-1, 2-2)and porB (3-82), all were null for nadA, and all had the same pulsedfield gel electrophoresis (PFGE) profile (429).

Genetic characterization of the 2013 University of California SantaBarbara Outbreak Strains is as follows: The UCSB strains were typed asCC32(ET5; ST32), expressed fHBP variant B24, and are related to theOregon clone that has been associated with hyperendemic serogroup Bdisease since 1993. Unlike the Princeton outbreak group of strains, theUCSB strains segregated genetically into two distinct clusters that weredifferentiated by their PFGE profile (468 or 467) and porB type (3-461or 3-24). The strains had identical allele assignments for NadA (1),NHBA (5), porA (subtype P1.7, 16-20)

hSBA titers at baseline for all subjects and all outbreak strains were<4, indicating that the subjects had no protective antibodies to any ofthe outbreak strains prior to immunization with bivalent rLP2086.

Results:

All 23 MnB strains were susceptible in hSBA with sera from individualsubjects immunized with bivalent rLP2086. Strains representing all 10prevalent fHBP variants as well as additional strains were all killed byhSBA. Baseline hSBA seroprotection rates (proportions of subjectsachieving hSBA titers ≧1:4) were generally low. The lower seroprotectiverates observed in subjects before immunization with bivalent rLP2086exemplify the vulnerability of a non-vaccinated adolescent or youngadult population to MnB disease. However, robust seroprotection rateswere observed in adolescents and young adults with postvaccination sera:seroprotection rates >70% were observed for 83% of these strainsdepending on MnB strains and population tested. Postvaccinationseroprotection rates for strains expressing the most prevalent subfamilyA and B fHBP variants, B24 and A22, ranged from 81.0% to 100%, and 77.8%to 100% for recent outbreak strains expressing fHBP variants B24 andB153. Furthermore, robust postdose 2 responses (compared to baseline) toall outbreak strains were observed in these subjects, ranging from 56 to89% depending on the outbreak strain used in the hSBA. In contrast,prevaccination seroprotective rates were low, or not detectable, forrecent US outbreak strains. The hSBA responses to the PrincetonUniversity and UCSB outbreak strains are shown in FIG. 2.

Conclusions:

Bivalent rLP2086 elicits robust seroprotective hSBA responses inindividuals to diverse invasive MnB strains expressing prevalent fHBPsin the US and Europe, as well as newly emerging variants (B153)(SEQ IDNO: 6). The proportion of subjects that showed a seroprotective responseafter immunization with bivalent rLP2086 greatly exceeded the proportionof subjects that was seroprotected at baseline. The data support thatbivalent rLP2086 has the potential to provide broad protection ofadolescents and young adults from invasive meningococcal serogroup Bdisease, including disease from recent outbreaks.

> B153 (SEQ ID NO: 6) CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGKAEEVAGSAEVKTVNGIRHIG LAAKQ

What is claimed is:
 1. A composition comprising a) a first lipidatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, b) a second lipidated polypeptide comprising the amino acid sequenceset forth in SEQ ID NO: 2,
 2. The composition according to claim 1,further comprising polysorbate-80, aluminum, histidine, and sodiumchloride.
 3. The composition according to claim 2, wherein thecomposition comprises about 120 μg/ml of the first polypeptide; about120 μg/ml of the second polypeptide; about 2.8 molar ratio ofpolysorbate-80; about 0.5 mg/ml aluminum; about 10 mM histidine; andabout 150 mM sodium chloride.
 4. The composition according to claim 2,wherein the composition comprises about 60 μg of the first polypeptide;about 60 μg of the second polypeptide; about 18 μg polysorbate-80; about250 μg aluminum; about 780 μg histidine; and about 4380 μg sodiumchloride.
 5. The composition according to claim 1, wherein thecomposition induces a bactericidal titer of serum immunoglobulin that isat least greater than 1-fold higher in the human after receiving thefirst dose than a bactericidal titer of serum immunoglobulin in thehuman prior to receiving the first dose, when measured under identicalconditions in a serum bactericidal assay using human complement.
 6. Thecomposition according to claim 1, wherein the composition induces abactericidal titer of serum immunoglobulin that is at least 2-foldhigher in the human after receiving the first dose than a bactericidaltiter of serum immunoglobulin in the human prior to receiving the firstdose, when measured under identical conditions in a serum bactericidalassay using human complement.
 7. The composition according to claim 1,wherein the composition induces a bactericidal titer of serumimmunoglobulin that is at least 4-fold higher in the human afterreceiving the first dose than a bactericidal titer of serumimmunoglobulin in the human prior to receiving the first dose, whenmeasured under identical conditions in a serum bactericidal assay usinghuman complement.
 8. The composition according to claim 1, wherein thecomposition induces a bactericidal titer of serum immunoglobulin that isat least 8-fold higher in the human after receiving the first dose thana bactericidal titer of serum immunoglobulin in the human prior toreceiving the first dose, when measured under identical conditions in aserum bactericidal assay using human complement.
 9. The compositionaccording to claim 1, wherein the composition does not further comprisea polypeptide having less than 100% sequence identity to SEQ ID NO: 1.10. The composition according to claim 1, wherein the first polypeptidehas a total of 258 amino acids.
 11. The composition according to claim1, wherein the first polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 3 at the N-terminus of the polypeptide.
 12. Thecomposition according to claim 1, wherein the composition does notfurther comprise a polypeptide having less than 100% sequence identityto SEQ ID NO:
 2. 13. The composition according to claim 1, wherein thesecond polypeptide has a total of 261 amino acids.
 14. The compositionaccording to claim 1, wherein the second polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 3 at the N-terminus of thepolypeptide.
 15. The composition according to claim 1, wherein thecomposition comprises at most two lipidated polypeptides.
 16. Thecomposition according to claim 1, wherein the composition does notfurther comprise a polypeptide having less than 100% sequence identityto SEQ ID NO:
 1. 17. The composition according to claim 1, wherein thecomposition does not further comprise a polypeptide having less than100% sequence identity to SEQ ID NO:
 2. 18. The composition according toclaim 1, wherein the composition does not comprise a hybrid protein. 19.The composition according to claim 1, wherein the composition does notcomprise a chimeric protein.
 20. The composition according to claim 1,wherein the composition does not comprise a fusion protein.
 21. Thecomposition according to claim 1, wherein the composition is notlyophilized.
 22. The composition according to claim 1, wherein thecomposition is a liquid composition.
 23. A method of inducing abactericidal immune response against a Neisseria meningitidis serogroupB subfamily A strain and against a Neisseria meningitidis serogroup Bsubfamily B strain in a human, comprising administering to the human aneffective amount of a composition, said composition comprising a) afirst lipidated polypeptide comprising the amino acid sequence set forthin SEQ ID NO: 1, and b) a second lipidated polypeptide comprising theamino acid sequence set forth in SEQ ID NO:
 2. 24. The method accordingto claim 23, wherein the composition further comprises polysorbate-80,aluminum, histidine, and sodium chloride.
 25. The method according toclaim 23, wherein the immune response against the Neisseria meningitidisserogroup B subfamily A strain is greater than the immune responseagainst the Neisseria meningitidis serogroup B subfamily B strain. 26.The method according to claim 23, wherein the immune response againstthe Neisseria meningitidis serogroup B subfamily A strain in the humancomprises a bactericidal titer that is greater than the bactericidaltiter against the Neisseria meningitidis serogroup B subfamily B strainin the human.
 27. The method according to claim 23, wherein thecomposition induces a bactericidal immune response against any one of N.meningitidis serogroup B A22, A56, B24, B44 strains, or any combinationthereof.
 28. The method according to claim 23, wherein the compositioninduces a bactericidal immune response against any one of N.meningitidis serogroup B B24, B16, B44, A22, B03, B09, A12, A19, A05,A07, B153 strains, or any combination thereof.
 29. The method accordingto claim 23, wherein the method further comprises administering to thehuman an immunogenic composition against human papillomavirus.
 30. Themethod according to claim 29, wherein the immunogenic compositionagainst human papillomavirus is administered to the human within 24hours of administering said composition against Neisseria meningitidis.31. The method according to claim 29, further comprising inducing animmune response against any one of human papillomavirus type 6, 11, 16,18, or any combination thereof.
 32. The method according to claim 23,wherein the method further comprises administering to the human animmunogenic composition against diphtheria, tetanus, pertussis andpoliomyelitis.
 33. The method according to claim 32, wherein theimmunogenic composition against diphtheria, tetanus, pertussis andpoliomyelitis is administered to the human within 24 hours ofadministering said composition against Neisseria meningitidis.
 34. Themethod according to claim 32, wherein the method further comprisesinducing an immune response against any one of diphtheria, tetanus,pertussis, poliomyelitis, or any combination thereof in the human.
 35. Amethod of inducing a bactericidal immune response against a Neisseriameningitidis serogroup B strain expressing B153 factor H binding proteinin a human, comprising administering to the human an effective amount ofa composition, said composition comprising a) a first lipidatedpolypeptide comprising the amino acid sequence set forth in SEQ ID NO:1, and b) a second lipidated polypeptide comprising the amino acidsequence set forth in SEQ ID NO: 2.